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Acta Biomaterialia 10 (2014) 630640

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Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actabiomat

Tunable hydrogel composite with two-step processing in combination with innovative hardware upgrade for cell-based three-dimensional bioprinting
Silke Wst, Marie E. Godla, Ralph Mller, Sandra Hofmann
Institute for Biomechanics, ETH Zurich, 8093 Zurich, Switzerland

a r t i c l e

i n f o

a b s t r a c t
Three-dimensional (3-D) bioprinting is the layer-by-layer deposition of biological material with the aim of achieving stable 3-D constructs for application in tissue engineering. It is a powerful tool for the spatially directed placement of multiple materials and/or cells within the 3-D sample. Encapsulated cells are protected by the bioink during the printing process. Very few materials are available that fulll requirements for bioprinting as well as provide adequate properties for cell encapsulation during and after the printing process. A hydrogel composite including alginate and gelatin precursors was tuned with different concentrations of hydroxyapatite (HA) and characterized in terms of rheology, swelling behavior and mechanical properties to assess the versatility of the system. Instantaneous as well as long-term structural integrity of the printed hydrogel was achieved with a two-step mechanism combining the thermosensitive properties of gelatin with chemical crosslinking of alginate. Novel syringe tip heaters were developed for improved temperature control of the bioink to avoid clogging. Human mesenchymal stem cells mixed into the hydrogel precursor survived the printing process and showed high cell viability of 85% living cells after 3 days of subsequent in vitro culture. HA enabled the visualization of the printed structures with micro-computed tomography. The inclusion of HA also favors the use of the bioink for bone tissue engineering applications. By adding factors other than HA, the composite could be used as a bioink for applications in drug delivery, microsphere deposition or soft tissue engineering. 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Article history: Received 12 July 2013 Received in revised form 27 September 2013 Accepted 15 October 2013 Available online 21 October 2013 Keywords: Cell-based 3-D bioprinting Hydrogel Two-step crosslinking Tissue engineering

1. Introduction Tissue engineering aims to rebuild a tissue by seeding cells onto a three-dimensional (3-D) scaffold and culture the construct in conjunction with biological signals [1]. Although this state-ofthe-art approach is being investigated widely, there are still key difculties to overcome. One issue is the lack of geometrical control during scaffold fabrication when using common methods such as salt-leaching [2], porogen melting [3] or gas foaming [4] and the associated difculties in up-scaling to clinically relevant sizes. Cell seeding is usually performed by pipetting cells by hand onto the scaffold, which results in inhomogeneous cell distribution and ingrowth into the scaffold [2]. A promising new method to overcome the current limitations is 3-D biofabrication, which is the application of rapid prototyping to tissue engineering, allowing layer-by-layer deposition of biological material [5]. 3-D bioprinting describes the deposition of the biomaterial itself. There are two methods of 3-D bioprinting: pure scaffold printing and 3-D tissue printing. The rst method focuses on
Corresponding author. Tel.: +41 44 633 70 82; fax: +41 44 633 15 73.
E-mail address: sahofmann@ethz.ch (S. Hofmann).

fabrication of scaffolds by printing pure scaffold material, aiming to control scaffold geometry or pore size. Post-treatment can be performed to enhance scaffold stability, remove cytotoxic crosslinkers or sterilize the scaffold before the addition of living cells. The second method refers to 3-D tissue printing which focuses on the deposition of biological material, where the bioink consists of scaffold material and biological components such as cells or growth factors simultaneously. Due to the presence of biological components this printing process requires each single step within the process to be biocompatible, providing gentle processing conditions. The great power of 3-D tissue printing is the multi-channel assembly of most commercial 3-D bioprinters. This enables the controlled deposition of different materials and/or different cell types adjacent to one another in a predened 3-D pattern within one process. Fabrication of soft cell-free hydrogel scaffolds has been widely performed with 3-D bioprinting [6,7]. So far there are only a few studies addressing the challenge of 3-D tissue printing cells into a spatially organized 3-D matrix [811]. In this study, we present a tunable hydrogel composite which provides a physiological environment for cells, mild processing conditions and results in a stable, viable 3-D construct.

1742-7061/$ - see front matter 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2013.10.016

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A suitable material for 3-D bioprinting of cells embedded in scaffold material must support cell viability and differentiation as well as be printable and maintain construct integrity and mechanical stability after printing. Physical stability requires that a liquid or semi-solid bioink can be chemically or physically crosslinked. Harsh conditions, such as extreme temperatures or pH and high shear stresses, while often necessary for the printing and crosslinking processes, must be avoided. These restrictions often result in a reduced printing resolution compared to the printing of scaffold material without cells. Biocompatible hydrogels show high potential for 3-D bioprinting due to their ability to induce a phase change from liquid to (semi-)solid by crosslinking. They have been widely used in soft tissue engineering and are lately considered as an emerging material for bone substitutes [12]. Alginate is one of the most used materials for cell-based hydrogel printing due to its favorable characteristics concerning biocompatibility and the capability to support cellular function and differentiation in culture, but also as a carrier for drug delivery [13]. Gelatin, which is also known to enhance cell attachment and promote cell growth [14,15], was implemented to improve the initial stability of the 3-D printed construct. Hydroxyapatite is known to be an excellent bone substitute material [16]. Enhancing alginate hydrogel with hydroxyapatite (HA) increases cell attachment [17] and leads to osteogenic differentiation of osteogenic progenitor cells in vivo [18]. The hydrogel composite presented in this study is one potential example for the presented two-step processing method. The materials here alginate and gelatin enhanced with varying amounts of HA have been specically selected for bone tissue engineering applications. A possible expansion of the presented processing method for other materials and for other applications will need detailed investigation. Our hypothesis is that combining gelatin and alginate improves instantaneous stability by maintaining initial bonding between single layers due to the gelation of gelatin as well as long-term stability of the whole construct by chemically crosslinking alginate. By adding HA, a versatile material is obtained which additionally leads to radiopacity of the material and thus visibility for X-ray-based micro-computed tomography (lCT), a powerful 3-D imaging technique to assess the printed construct. In this study we assessed the inuence of varying amounts of HA on material properties, cell viability after printing and its impact on the printing process. Cells were mixed into the hydrogel precursor before printing and stabilized in their 3-D position once the composite was crosslinked. New hardware consisting of a heatable print head with innovative syringe tip heaters was manufactured to adapt a commercially available 3-D bioprinter for this process.

or no HA, 2% (w/v) alginate and 10% (w/v) gelatin, respectively. Accordingly, the three different groups can be labeled: hydrogel 8% HA, hydrogel 4% HA and hydrogel no HA. Hydrogels were pasteurized at 72 C for 1 h for all experiments. 2.2. 3-D bioprinting system The schematic printing process is depicted in Fig. 1. An opensource two-syringe Fab@Home 3-D printer Model 2 (The Nextfab Store, Albuquerque, NM, USA) was modied with an in-housefabricated heatable print-head to induce the thermo-sensitive behavior of the hydrogel (Fig. 2A and D). This print-head was designed to precisely t two standard 10 ml syringes with luer lock connections (Becton Dickinson AG, Allschwil, Switzerland) and was equipped with a syringe heating pad and a temperature controller unit (New Era Pump Systems, Inc., Farmingdale, NY, USA). A unique feature of the heatable print-head is the two dispense tip metal caps, which surround the dispense tips and transfer heat from the heating pad down to the end of the tip (Fig. 2B and D). Coordinate-based printing paths were created digitally and transferred to the printer electronically. Hydrogel was heated up to 40 C to liquify, loaded into syringes and mounted to the print head set at 40 C. The substrate was cooled to 10 C, which led to solidication and stabilization of the printed hydrogel (Fig. 2C). 2.3. Material characterization Hydroxyapatite powder used for the hydrogel composites was characterized with powder X-ray diffraction on a CubiX diffractometer (PANalytical, Almelo, The Netherlands) to determine the crystalline phase composition (Fig. S.1). Specic surface area (SSA) was analyzed using the BrunauerEmmettTeller model (Gemini 2360, Micromeritics, USA) and scanning electron microscopy (SEM) was performed with an EVO MA 25 (Zeiss, Germany) to assess the morphology of the used HA powder (Fig. S.2). The hydrogel composites were investigated regarding viscosity, mechanical properties and hydrogel swelling. For the assessment of pure material properties, objects were molded to avoid inuence of structure and surface due to lament deposition. Hydrogels were monitored during 3 days, assessing instantaneous situation, equilibrium and a trend for long-term culture. Hydrogel discs with homogeneous bulk material were used for the assessment of swelling and mechanical properties. They were prepared by pouring liquid hydrogel (at 40 C) into molds of 5 mm diameter and 2 mm height onto a substrate cooled to 10 C. Once removed from the mold, samples were crosslinked in 2% calcium chloride (CaCl2) (w/v) (VWR, Dietikon, Switzerland) in PBS for 10 min before heating the discs to 21 C (room temperature) or 37 C (incubator temperature) or allowing them to dry at room temperature, depending on the experimental setup. 2.3.1. Rheology Rheological properties of the three different hydrogels (n = 5 per group) were measured using a HAAKE RheoStress 600 rheometer (thermo Electron GmbH, Karlsruhe, Germany) using a cone-plate measuring geometry with a diameter of 35 mm, a cone-to-plate distance of 105 lm and a cone angle of 2. A solid sample of 400 ll (425430 mg) hydrogel was placed on the plate and melted at 42 C to completely ll the gap between the rotating cone and the stationary at plate. To avoid water evaporation during the tests, 1 ml PBS was distributed next to the sample on the plate and the whole setup was covered with a jacket. Viscosity and shear stress were measured at 37 C with a rotatory test setup by varying the shear rate from 0.0001 s1 to 100 s1 and back during 400 s. Storage modulus (G0 ) and loss modulus (G00 ) were assessed by oscil-

2. Materials and methods 2.1. Hydrogel precursor fabrication Three different hydrogel compositions with varying amounts of HA were prepared: pure HA powder (Acros Organics, Geel, Belgium) (16%, 8% and 0% w/v) was mixed in phosphate buffered saline (PBS) (Medicago, Uppsala, Sweden), forming a homogeneous suspension. Low viscosity alginic acid (4% w/v) derived from brown algae (macrocystis pyrifera) with a G- to M-unit ratio of 0.39 (MW: 12,00080,000) (Sigma Aldrich, Buchs, Switzerland) was gradually added to the HAPBS suspension for 10 min under constant stirring and further stirred for 2 h. Type A gelatin (20% w/v) from porcine skin (MW: 50,000100,000) (Sigma Aldrich, Buchs, Switzerland) was dissolved in PBS and kept 2 h at 60 C to homogenize and dissolve remaining air bubbles. The alginateHAPBS solution was thoroughly mixed with the gelatinPBS solution in a 1:1 ratio, leading to nal concentrations of 8% (w/v), 4% (w/v)

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Underlying mechanism:
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Ca2+

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Fig. 1. Schematics of the two-step hydrogel gelation mechanism based on reversible thermal gelation of gelatin and irreversible chemical gelation of alginate on the polymer level. (A) Printing of heated hydrogel precursor including living cells onto a cold substrate. (B) Instantaneous gelation and primary strand stability takes place due to the temperature drop, which leads to solidication of the gelatin. (C) Once the whole construct is printed it is immersed in a CaCl2 bath to crosslink the alginate present in the hydrogel precursor. This procedure takes place in a cold environment to maintain construct stability until chemical crosslinking is completed. (D) Long-term stability is ensured by the crosslinked alginate and the cooling plate can be removed.

syringe heater

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Fig. 2. Details of the Fab@Home printer hardware modications: (A) heating pad attached to the in-house designed syringe heater, (B) syringe tip heater plugged into the syringe heater and tightened with a xation device, (C) printed hydrogel lament on the cooled substrate and (D) design layout of the new syringe and tip heater together with the two syringes which can contain two different hydrogel composites/cell types for printing.

latory tests over a temperature ramp from 50 C to 10 C and back to 50 C. The rate of temperature change was 1 C min1, the oscillation frequency was 1 Hz and the constant shear amplitude was 50 Pa. The gel point describes the temperature of a material when it solidies. It is dened as the intersection of the storage modulus G0 curve and the loss modulus G00 curve during temperature decrease at an oscillatory measurement.

2.3.2. Swelling Hydrogel swelling was assessed by measuring the weight of hydrogel discs over the rst 3 days after immersion in PBS containing 0.25% CaCl2. Three different experimental setups were analyzed with the three different hydrogel composites (n = 5 per group). In the rst setup, the hydrogel discs were prepared and air-dried. Swelling at 21 C was measured from when the dry hydrogel discs

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were rst immersed. For the second setup, hydrogel discs were freshly prepared and swelling assessments at 21 C were initiated immediately after crosslinking. Samples for the third setup were also freshly prepared but kept at 37 C during the experiment. The different setups reect the classical hydrogel swelling assessment at room temperature (setup 1), the applied experimental conditions during 3-D bioprinting with subsequent culture at 37 C (setup 3) and an additional setup facilitating comparison between setups 1 and 3 (setup 2). Swelling is reported as ratio of hydrogel weight over time with respect to the initial hydrogel weight, which is dry weight for setup 1 and wet weight for setups 2 and 3. 2.3.3. Mechanical properties Youngs modulus and maximal force were assessed on a Zwick material testing machine (Zwick 1456, Ulm, Germany) with a 10 N load cell at room temperature. The preload of 5 mN was approached with 5 mm min1 and unconned uniaxial compression tests were performed in a position controlled fashion with a strain rate of 1 mm min1 until a 60% deformation of the construct was reached. Freshly prepared hydrogel discs from the three hydrogel composites (n = 5 per group) were measured during 3 consecutive days at room temperature. Samples were stored in PBS containing 0.25% CaCl2 between the measurements; no loss of hydration was assumed during the 5 min total measurement time per sample and day. The Youngs modulus was calculated from the linear region of the stressstrain curve, which was between 5% and 15% deformation for all samples at all conditions. Maximal deformation before sample break occurred was found to be 60%. This value was chosen as parameter for force measurements. 2.4. Hydrogel printing: single layer analysis Hydrogel laments in a spiral structure were printed with the three different hydrogel composites to characterize the minimal distance between adjacent strands until fusion of strands takes place. The tested distances between laments were 2 mm, 1 mm and 500 lm. A 27 gauge plastic dispense tip (EFD Nordson, Vilters, Switzerland), which corresponds to an inner tip diameter of 200 lm, was used. Printing parameters were set to a path speed of 2 mm s-1 and a deposition rate of 0.45 103 mm mm1 (mm piston motion per mm travel). Visualization of the printed structures was performed with a stereomicroscope and lCT imaging. 2.4.1. Stereomicroscope Two-dimensional top view images of hydrogel spirals were taken with a stereomicroscope (Wild M8, Leica Microsystems, Heerbrugg, Switzerland) directly after printing. 2.4.2. lCT imaging Samples were xed after stereomicroscope imaging in 10% buffered formalin (Sigma Aldrich, Buchs, Switzerland) for 24 h at room temperature. Formalin was removed, samples were washed in PBS, PBS droplets were carefully wiped to remove the remaining PBS around the laments and samples were mounted in a lCT vial. lCT imaging was performed on a lCT 40 scanner (Scanco Medical AG, Brttisellen, Switzerland) with an isotropic nominal resolution of 10 lm. The energy of the X-ray tube was 45 kVp, the intensity was 177 lA, the integration time was set to 300 ms and twofold frame averaging was used. A constrained Gaussian lter was applied to the resulting images with a lter width of 3 and lter support of 3. Segmentation was performed using a global threshold of 80 for hydrogel 8% HA and hydrogel 4% HA, and a global threshold of 70 was needed to visualize hydrogel no HA.

2.5. Cell printing: 3 day viability study A cell experiment was performed to investigate cell compatibility of the introduced new hydrogel composites in combination with the printing and two-step post-processing treatment. The printer was moved into the biosafety cabinet providing aseptic conditions for the experiment. The three different hydrogels (n = 5 per group) were enhanced with cells and the same three hydrogels (n = 5 per group) without cells were used as control groups. Human mesenchymal stem cells (hMSCs) in fth passage (P5) as described in Ref. [19] were mixed into the hydrogel precursor preheated to 40 C at a concentration of 5 million cells per ml and thoroughly mixed with a spatula. A sterile syringe was lled with the solution and mounted in the print head. A cube consisting of eight layers, each with a surface area of 8 8 mm2, was printed onto a cold substrate with a 25 gauge plastic dispense tip (EFD Nordson, Vilters, Switzerland), which corresponds to an inner tip diameter of 250 lm. The path speed of the print head was 2 mm s1, deposition rate 2 103 mm mm1 and layer height 300 lm. Samples were crosslinked with 2% (w/v) CaCl2 (VWR, Dietikon, Switzerland) in PBS (Medicago, Uppsala, Sweden) for 10 min after printing. Control medium comprising Dulbeccos modied Eagles medium (DMEM) with 10% fetal bovine serum (FBS), and 1% antibiotic/antimycotic solution consisting of 10000 units ml1 penicillin, 10000 lg ml1 streptomycin and 25 lg ml1 Fungizone (amphotericin B) (all substances obtained from Invitrogen, Basel, Switzerland) was supplemented with 0.25% (w/v) CaCl2 (VWR, Dietikon, Switzerland) to enhance and maintain the structural integrity of the scaffold during 3 days of in vitro culture. 2.5.1. Cell viability analysis Cell viability of all samples was assessed with a LIVE/DEAD Viability/Cytotoxicity assay from Molecular Probes (Life Technologies Ltd., Zug, Switzerland) 3 days after printing. Samples were washed in PBS and a 1 mm thick slice was cut out along the cross-section in the middle of the sample (Fig. 7A). Constructs were transferred to a 2 lM Calcein AM4 lM ethidium homodimer (EthD-1) solution for 30 min before imaging with a confocal microscope (Olympus 5B04). In this protocol, living cells are stained with a uorescent marker Calcein AM and dead cells with EthD-1, resulting in living cells being displayed in green using an argon laser (488 nm) and Alexa Fluor 488 dye and dead cells being displayed in red with the HeNe laser (543 nm) and propidium iodide dye. Living and dead cells were counted manually by the same operator in three images per group and averaged. 2.6. Two-phase printing One major application of 3-D bioprinting for tissue engineering is to be able to place different materials or cells next to each other in predened patterns, where spatial limitations are difcult or impossible to overcome when constructs are generated by hand. A two-phase construct was designed with three channels, each 500 lm in diameter over a length of 10 mm. The bulk phase consisted of hydrogel 8% HA and the tube phase of hydrogel no HA. A 27 gauge plastic dispense tip (EFD Nordson, Vilters, Switzerland) with an inner tip diameter of 200 lm was used; path speed was set to 2 mm s1, deposition rate to 0.8 103 mm mm1 and layer height to 350 lm. Printed samples were crosslinked with 2% (w/ v) CaCl2 in PBS for 10 min, xed in 10% buffered formalin for 24 h at room temperature, formalin was removed and PBS was added to samples. lCT imaging was used to evaluate samples as described in Section 2.4.2. X-ray absorption of the two phases was different due to differences in HA content; phases were separated with a global threshold of 130.

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2.7. Statistical analysis All quantitative values are reported as means standard deviation with n = 5 samples per group. Statistical analysis of variance (ANOVA) was performed using PASW Statistics 18 (SPSS Inc., Chicago, IL, USA). To determine the gel point of the hydrogels univariate ANOVA Bonferroni post hoc with Bonferroni correction was used. Statistical differences in swelling ratios normalized to day 0 were analyzed at day 1 and day 3 with a univariate analysis of variance followed by Students t-tests and Bonferroni correction for multiple comparisons. Mechanical properties were examined at day 0, day 1 and day 3 with a univariate ANOVA and Bonferroni correction for multiple comparisons. P-values <0.05 were considered statistically signicant, P-values <0.01 were considered highly statistically signicant.

subsequent cell culture the construct was placed in an incubator at 37 C, which leads to a temperature rise above the gel point of the hydrogel (Fig. 1D). Gelatin acts as a short term stabilizer that is dissolved after some time at 37 C. Since long-term stability is provided by alginate the loss of gelatin has no impact on the geometry. 3.2. Printer hardware adaptation The Fab@Home 3-D printer used for the study is commercially available and, as an open-source package, is easy to adapt to specic applications. For the herein presented thermo-sensitive hydrogel a temperature-controlled print-head was designed and fabricated in-house (Fig. 2A and D). To improve heating performance, special focus was put on the development of a feature which allows heating of the dispense tips. The tip heater consists of a metal cap which surrounds the dispense tips and transfers the heat from the heating pad down to the end of the tip (Fig. 2B and D). With the hardware adaptation, consistent performance of the hydrogel was achieved and clogging of the dispense tips was avoided (Fig. 2C). 3.3. Hydrogel characterization The rheological behavior, swelling properties and mechanical stiffness of alginategelatin hydrogels enhanced with varying amounts of HA (8%, 4% and 0%) were analyzed. 3.3.1. Viscosity Viscosity affects the printing process primarily at the dispense tip, the most critical location during extrusion. If viscosity is too high the hydrogel will clog and the printing process cannot be continued. As expected, viscosity was highest for the hydrogel 8% HA and linearly decreased with lower HA concentrations (Fig. 3A). With a shear rate of 0.0001 s1 the viscosity was 5.6 Pa s for

3. Results 3.1. Hydrogel development The unique stability of the 3-D construct during and after printing is a result of the combination of physical and chemical crosslinking the alginategelatinHA hydrogel. Instantaneous stability, which maintains the shape of the printed hydrogel lament and leads to a more accurate structure, was achieved by adding gelatin to the commonly used alginate hydrogel. A schematic of the process is shown in Fig. 1. The composite is liquid at 40 C but solidies instantaneously once it is cooled below 10 C due to the temperature-dependent hysteresis behavior of gelatin (Fig. 1A and B). Alginate solidies when crosslinked with CaCl2. This chemical process is slower than the temperature-dependent physical solidication and requires several minutes. Chemical crosslinking must occur after printing while the whole construct is still below the gel point to maintain stability of the geometry (Fig. 1C). For

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Fig. 3. Rheological properties of the three hydrogel composites: hydrogel 8% HA, hydrogel 4% HA and hydrogel no HA. (A) Viscosity and (B) shear stress is plotted over a shear rate from 0.0001 to 100 s1. Shear stress curves show shear thinning behavior of the hydrogels. (C) Representative image of hydrogel 8% HA group showing the determination of the gel point, which is dened as the intersection of storage modulus G0 and loss modulus G00 during cooling from 50 C to 10 C. (D) Average gel point of the three different groups (n = 5). P < 0.05, P < 0.01.

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hydrogel 8% HA, 3.4 Pa s for hydrogel 4% HA and 2.3 Pa s for hydrogel no HA. The viscosity decreased with increasing shear rates for all three composites. Shear stress (Fig. 3B) increased with shear rate for all three hydrogels. Shear stress at a shear rate of 100 s1 was 220 Pa for hydrogel 8% HA, 170 Pa for hydrogel 4% HA and 110 Pa for hydrogel no HA. The shape of the shear stress curve characterizes the hydrogel composite as a shear thinning material. Representative curves of G0 and G00 of a sample of the hydrogel 8% HA group is shown in Fig. 3C. The inuence of the amount of HA on the gel point is shown in Fig. 3D. For hydrogel 8% HA the gel point was 28.2 0.0 C, for hydrogel 4% HA it was 27.8 0.2 C and for hydrogel no HA 25.6 0.2 C. There was a highly statistically signicant difference (P < 0.01) between composites with and without HA. The difference between the two groups with different amounts of HA was also signicant (P < 0.05). 3.3.2. Swelling Swelling was investigated under three different experimental conditions: (i) swelling at 21 C starting with immersion of dried hydrogel discs in solution (Fig. 4A); (ii) immersion of freshly prepared hydrogel discs (Fig. 4B); and (iii) swelling at 37 C with freshly prepared hydrogel discs (Fig. 4C). Swelling is reported as ra-

tio Q of the hydrogel weight with respect to the initial hydrogel weight. Swelling at 21 C led to an increase in hydrogel weight caused by PBS uptake of the hydrogel, with a 5- to 8-fold increase started from dried hydrogel discs (Fig. 4A) and a 1.3- to 1.6-fold increase started from fresh hydrogel discs (Fig. 4B). Experimental conditions at 37 C led to a short peak in swelling after 3 h up to a factor of 1.6, but over longer time hydrogel weight dropped to 8090% of the original weight. For all hydrogels and experimental conditions equilibrium was reached after 24 h. Swelling behavior of the three hydrogels was similar within the same experimental condition. Starting with dry hydrogel discs at 21 C the hydrogel no HA group swelled most compared to the hydrogels containing HA, and hydrogel 8% HA swelling was highly statistically signicantly less (P < 0.01) than the other groups. On the opposite, starting with fresh hydrogel discs at 21 C, the hydrogel no HA group swelled least compared to the groups containing HA, with a highly statistically signicant difference to the hydrogel 4% HA group. Experimental conditions at 37 C revealed an increase in swelling for all hydrogels in the rst 3 h followed by a decrease in swelling ratio. The least swelling and least degradation was found for the hydrogel without HA and was highly statistically signicantly higher (P < 0.01) for both HA-containing groups. 3.3.3. Mechanical properties Stressstrain curves of representative samples from hydrogel 8% HA, hydrogel 4% HA and hydrogel no HA at day 3 is shown in Fig. 5AC. Mechanical properties were investigated in terms of Youngs modulus (E) (Fig. 5D) and the maximal force at 60% deformation (Fmax(e=60%)) (Fig. 5E). The three hydrogels were tested over 3 days, to assess the material property right after printing (day 0), at equilibrium of swelling (day 1) and after 3 days in culture. For all groups the Youngs modulus increase was highly statistically signicant (P < 0.01) between day 0 and day 1 after printing and the decrease was highly statistically signicant (P < 0.01) between day 1 and day 3. At day 0 the amount of HA in the hydrogel correlated with the stiffness of the hydrogel disc, with 36 3 kPa for hydrogel 8% HA, 32 2 kPa for hydrogel 4% HA and 29 2 kPa for hydrogel no HA. The Youngs modulus change was highly statistically signicantly over time (P < 0.01) as was the correlation between the amount of HA and stiffness. At day 1 Youngs modulus was 37 1 kPa for hydrogel 8% HA, 33 1 kPa for hydrogel 4% HA and 37 1 kPa for hydrogel no HA and after 3 days 32 2 kPa, 30 1 kPa and 32 1 kPa, respectively. Hydrogel 8% HA was highly statistically signicantly different from hydrogel 4% HA (P < 0.01) and statistically signicantly different from hydrogel no HA (P < 0.05); no statistical difference was detected between hydrogel 4% HA and hydrogel no HA. Concerning the maximal force at 60% deformation of the hydrogel disc, the behavior of the three hydrogels was similar over time, with a highly statistically signicant difference (P < 0.01) between hydrogel 8% HA and hydrogel 4% HA, hydrogel 8% HA and hydrogel no HA and hydrogel 4% HA and hydrogel no HA. The highest Fmax was measured for hydrogel no HA for all time points, with 1.64 0.05 N at day 0, followed by hydrogel 8% HA with 1.51 0.08 N and hydrogel 4% HA with 1.42 0.03 N. After 1 day, Fmax was statistically signicantly lower (P < 0.05) with 1.54 0.07 N for hydrogel no HA, 1.46 0.05 N for hydrogel 8% HA and 1.33 0.03 N for hydrogel 4% HA. After 3 days Fmax was highly statistically signicant higher (P < 0.01) compared to day 1 to 1.65 0.13 N, 1.57 0.02 N and 1.46 0.04 N, respectively. 3.4. Hydrogel printing: single layer analysis Hydrogel laments in a spiral structure were printed to investigate the minimal distance between strands before fusion of adjacent laments take place. The top view of printed spirals of the

hydrogel swelling Q [w(t)/winitial]

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Fig. 4. Hydrogel swelling of the three composites over 3 days at different experimental conditions: (A) swelling at 21 C after immersion of dried hydrogel discs in PBS containing 0.25% CaCl2, (B) swelling at 21 C started with immersion of freshly prepared hydrogel discs in PBS containing 0.25% CaCl2 and (C) swelling at 37 C started with immersion of freshly prepared hydrogel discs (n = 5) in PBS containing 0.25% CaCl2. Swelling is reported as ratio of hydrogel weight over time compared to the initial hydrogel weight; which is dry weight for (A) and wet weight for (B and C). Standard deviation was plotted in one direction only for better visualization. P < 0.05, P < 0.01.

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Fig. 5. (AC) Stressstrain curves of representative samples from hydrogel 8% HA, hydrogel 4% HA and hydrogel no HA at day 3. (D and E) Compressive mechanical properties of hydrogel discs of the three hydrogel composites over 3 days. (D) Youngs modulus at day 0 (right after printing), day 1 (at hydrogel equilibrium) and day 3. (E) Maximal force at 60% deformation at day 0, day 1 and day 3. Standard deviation was plotted in one direction only for better visualization. P < 0.05, P < 0.01.

three hydrogels was visualized with a stereomicroscope (Fig. 6A) and lCT imaging (Fig. 6B) and a slice in the xy plane (Fig. 6C). For all three hydrogel compositions the minimal distance without material fusion was 1 mm whereas fusion took place at strand distance of 500 lm. With higher amounts of HA and therefore higher viscosity of the hydrogel, printed laments tended to become more inhomogeneous and hydrogel accumulation in the corners was detected. As expected, superior contrast in lCT was obtained with hydrogels containing HA.

The printed construct was evaluated with lCT. Fig. 8 shows three different views of the lCT image: an overview of the sample showing mainly bulk material and the entrance of the tubes (Fig. 8A), a cross-sectional view at the location of the tubes within the bulk (Fig. 8B) and a negative image focusing on the tubular structure without surrounding bulk (Fig. 8A). The images reveal a stable 3D object consisting of two phases that were physically connected at the interface and could be accurately separated with an appropriate threshold with lCT. 4. Discussion Controlled spatial cell deposition within a biomaterial is a unique strength of 3-D bioprinting to create fully engineered tissue. First studies in 3-D bioplotting have shown that cells survive the printing process and are also able to keep their biological function [11,20,21]. Cells have the capacity to self-organize and self-assemble, forming tissue when the right external conditions are given [15,22]. Today, most biocompatible hydrogels available on the market or used in research either lack the appropriate mechanical properties for stability after printing or maintain structural integrity only by using high cure temperatures or cytotoxic solvents. Developing a biomaterial suitable for precise cell printing is one of the most challenging impediments towards 3-D tissue printing. Such a material must protect cells and other included biological factors from damage or drying during processing and provide a stimulatory environment after printing to allow cells to attach, proliferate and/or differentiate to eventually produce their own

3.5. Cell printing: 3 day viability study Representative uorescence images of the three groups containing cells after 3 days in culture are shown in Fig. 7B. Living cells appear in green and dead cells are shown in red. A majority of living cells were detected in all three hydrogel composites, with 15.2 9.7% dead cells in the hydrogel 8% HA group, 16.1 5.7% dead cells in the hydrogel 4% HA group and 15.4 5.9% dead cells in the hydrogel no HA group. There was no statistically signicant difference between the groups. Control groups without cells did not show any staining.

3.6. Two-phase printing A geometry with two phases was designed with a HA-containing phase representing bone tissue in combination with a alginate gelatin-lled tubular structure representing a vascular structure.

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hydrogel 8% HA

hydrogel 4% HA

hydrogel no HA

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Fig. 6. Hydrogel laments of the three different hydrogels were printed in spiral structure. (A) Top view images of printed structures acquired by stereomicroscope; (B and C) 3-D analysis of the different hydrogel composites in top view (B) and a slice in the xy plane (C) by lCT.

1mm

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100 m

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hydrogel 4% HA

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Fig. 7. Cell viability of bioprinted hMSCs encapsulated in hydrogel. (A) Experimental setup indicating size of the printed construct and the slice cut. (B) Representative uorescent images of labeled cells encapsulated in the three different hydrogels at day 3 after printing taken with a confocal microscope. Living cells are depicted in green and dead cells in red (dead cells highlighted with arrows). Control groups contained no cells and did not show any live or dead cells.

matrix. The material must be liquid in the print head to avoid clogging of the tip, but must also be solid once in contact with the substrate to take on and maintain the desired geometry in the deposition process and avoid any loss of structural integrity. With the hydrogel composite and the adapted two-step crosslinking process presented here, we have been able to address all of these requirements.

Printing of alginate hydrogels without gelatin into a crosslinker solution resulted in stable single laments, but there was no attachment to the substrate and also no binding between additional layers (data not shown). Additionally, cells were variably exposed to the crosslinker, with cells in the rst layers exposed much longer than cells in the later layers. If the whole construct was crosslinked only after printing, blurring of the material and move-

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1mm

Fig. 8. Two-phase printing of tubular structures within a 3-D hydrogel construct. Sample was visualized with uCT. (A) Side view of the whole sample, (B) crosssectional view revealing the tubes inside the construct and (C) visualization of void volume for a 3-D view of the tubular structures.

ment of the interface when processing two-phase constructs took place during printing and the construct resulted in an unstable and inaccurate shape (data not shown). It was hypothesized that these limitations could be overcome and construct stability increased by adding gelatin to the alginate hydrogel precursor and applying a two-step gelation process. When the preheated hydrogel precursor came into contact with the cold substrate during printing it induced immediate gelation due to the thermoreversible gelatin and the geometry of the single strands were maintained (Fig. 1A and B). This was followed by chemical crosslinking of alginate after the whole construct was printed (Fig. 1C and D). Chemical crosslinking of alginate is much slower and irreversible compared to physical crosslinking of gelatin. Nevertheless, it is stable at physiological culture conditions and important for long-term stability. When the construct was kept at 10 C until alginate crosslinking was completed there was no loss in structural stability during the whole process. Hydroxyapatite is known to be a suitable bone substitute material due to its biocompatibility, osteoconductivity and its similarity to the inorganic phase in natural bone [16]. With HA the alginate gelatin composite can be modied to increase the range of applications. The reasons for incorporating HA was its osteogenicity [16 18] and the possibility to use it as a contrast agent to visualize and characterize the printed hydrogel in 3-D with lCT. Visualization of a hydrogel with lCT is possible when it is contrasted against a gas phase (air) (Fig. 6B). However, the contrast is typically very weak and it is almost impossible to assess the complete structure due to noise and other imaging artifacts. When cells are incorporated, the physiological environment will be an aqueous phase, where either the hydrogel or its surrounding needs to be enhanced with a contrast agent for visualization. The two-phase 3-D construct (Fig. 8) shows that lCT is a powerful technique to separate and analyze different phases both qualitatively and quantitatively within one construct provided that materials with different absorption coefcients are used, which is the case here due to

the incorporation of HA. A clear separation of the two phases with lCT can be achieved by segmenting the image with an appropriate threshold. Physically, the two phases are connected at the interface and cannot fall or move apart. With similar material properties of the two phases the same mechanism for the binding between adjacent strands and layers of the same phase occurs at the interface due to the two-step crosslinking procedure. Additionally, due to the phase separation, scaffold degradation can be monitored with lCT when HA is incorporated. Another reason for the incorporation of HA to the hydrogel was to improve mechanical stiffness of the hydrogels. Mechanical properties are of interest since it has been shown that cells differentiate according to matrix stiffness and environment [23]. In contrast to what was expected, varying HA concentrations did not lead to large differences in the Youngs modulus and the maximal force at 60% deformation of the composites (Fig. 5). In contrast to our ndings, Lin and Yeh [17] varied HA concentration between 0% and 50% in an alginate scaffold and detected enhanced compressive strength of the composite with increasing HA content. A possible explanation for the different outcomes could be the much higher amount of HA loading in Lin and Yehs study compared to the maximum of 8% HA in this study. It could also be due to the fabrication procedure; Lin and Yeh freeze-dried the alginateHA gel solution after molding before the tests were performed. Freeze-drying leads to a different texture and mechanical properties of the hydrogel compared to wet hydrogel discs which were used directly after preparation in our study; this could alter the inuence of HA on the material stiffness. The other difference of Lin and Yehs study compared to our setup is the gelatin component, which was additionally included in the material presented in this study. By looking at the mechanical behavior of the hydrogels over time, the expectations were met. At room temperature a constant hydrogel weight to initial hydrogel weight ratio was detected after reaching the swelling equilibrium. Due to this constant ratio no large change in mechanical properties was assumed, after the point of swelling equilibrium was reached. The Youngs modulus of bone is in the MPa to GPa range, which cannot be achieved directly with a hydrogel construct. Encapsulated hMSCs are thought to produce their own extracellular matrix, which eventually mineralizes when the right extrinsic signals are given [24]. The goal to enhance alginategelatin with HA was to provide a suitable environment for the cells. Mechanical integrity of the constructs and thus a higher Youngs modulus will be achieved with the mineralized extracellular matrix. Introducing HA into the alginategelatin composite rendered the solution more viscous and more difcult to handle and print. Maximal HA inclusion to ensure proper handling and printing was found to be limited to 8%. With 8% HA the minimal distance before fusion of adjacent strands took place was still 1 mm even though the laments were slightly more irregular and also more hydrogel was deposited in corners of printed objects, as seen in single layer printing (Fig. 6). The hydrogel composition in this study was balanced between the optimal amount of alginate inuencing long-term stability; the concentration of gelatin, which is responsible for the instantaneous scaffold geometry stability; and the amount of HA with respect to the limits on viscosity of the printing process. The work of Tian et al. [7], who also investigated ow behavior of alginateHA composites with varying amounts of alginate and HA corroborates our results with an alginate concentration of 2%. The proposed system combines the good cell viability of alginate and the enhanced scaffold stability of gelatin. Together with osteoinductive HA, we achieved an improved cell-based 3-D construct for bone tissue engineering. The hardware of the 3-D bioprinter had to be modied to address the temperature-dependent gelation of gelatin as instantaneous stabilizer (Fig. 2). The heatable print-head and the

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additional tip heaters allowed homogeneous deposition of the hydrogel. Clogging of the dispense dips, which was a problem when using only the heatable print-head, especially when printing two phases within one construct, was solved with the tip heaters. Due to the tip heaters maintaining the liquidity of the bioink, the printing substrate could be cooled down enough to guarantee the instantaneous gelation of gelatin without hydrogel clogging and thus a stable 3-D construct that maintained the shape of the predened geometry. Viscosity is relevant during printing to assess the forces created due to shear stress at material extrusion. These forces act on cells when encapsulated. If viscosity is too high, clogging of the tip and inhomogeneous strand deposition can be a serious limitation. The gel point describes the temperature of a material when it solidies. Gel points of the hydrogels including HA were higher than the hydrogel no HA group, which means that the instantaneous stability is achieved at higher temperatures (Fig. 3D). The gel points of all three composites are above room temperature, which would theoretically not require any cooling on the substrate to induce gelation since printing takes place at room temperature. From former experiments it was observed that gelation is much faster when the substrate is additionally cooled down to 10 C, which is at the equilibrium of the storage modulus curve (Fig. 3C); this leads to improved structural stability of the geometry (data not shown). This is in accordance with the physical mechanism; when the hydrogel precursor heated up to 40 C is printed on a substrate kept at room temperature, the temperature gradient is smaller compared to when it is printed at a substrate cooled to 10 C. With a higher gradient the temperature in the middle of the printed strand is cooled down faster and thus the time to reach the gel point in the middle of the strand is shorter. Hydrogels are well known for their swelling behavior, which can be completely different depending on the material and is not predictable in composites. Hydrogel swelling does not only inuence the outer geometry of the constructs; it also has an inuence on the material properties itself (Fig. 5). Swelling is important to consider in 3-D bioprinting since it changes the size of the printed laments and consequently the whole construct in culture. Once the swelling properties are known, behavior of the printed geometry can be predicted. Swelling behavior needs to be accounted for initially in the design process. For instance, when hollow structures are printed within a construct, they must be designed such that the required size is reached at the equilibrium state of the material to avoid lling of the void space due to swelling. Equal accounting has to be done if one of the phases is designed to be dissolved immediately after the printing process. The two-phase construct presented in Fig. 8 was designed with coexistent phases of hydrogel with 8% and hydrogel without HA. It led to an increase in the whole construct during swelling. Looking at the swelling properties of the two hydrogels, the experimental condition of the printed twophase construct is in accordance with the freshly prepared hydrogels which were kept at 21 C after processing (Fig. 4B). No statistical difference in swelling between hydrogel 8% HA and hydrogel no HA was detected. Due to the same swelling properties of the two phases separately a homogenous swelling of the whole construct was assumed. Hydrogel degradation is another factor that affects cell culture. At culture conditions of 37 C, the hydrogel degradation starts earlier compared to culture at room temperature and signicantly inuenced hydrogel swelling after 3 h (Fig. 4C). From the behavior of storage and loss modulus (Fig. 3D) it is known that the hydrogel precursor becomes liquid at 37 C during heating. Due to the thermoreversibility of gelatin a similar effect happens during the swelling process of the chemically crosslinked hydrogel discs at 37 C, with the difference of alginate being irreversibly crosslinked. The liquid state of gelatin at 37 C acceler-

ates hydrogel degradation, while not much change is detected for samples at room temperature where gelatin is still in a solid state. The most critical factor for bioprinting is the capability of the bulk material to protect cells and/or other potential biological components such as growth factors or proteins during printing from high shear stresses, maintain a highly hydrated environment to avoid drying and provide a physiological environment after printing. This is extremely important for encapsulated cells but also for other applications such as drug delivery, where the activity of sensitive drugs needs to be guaranteed. Cell viability analysis did not reveal any differences of hMSCs encapsulated in the three different hydrogel composites over 3 days of in vitro culture. For all three hydrogels an average cell viability of 8485% 3 days after printing was achieved, which is a good result for bioprinting of hMSCs. For example, Fedorovich and coworkers had a cell viability of MSCs in alginate of between 60% and 85% 24 h after printing [18]. The inclusion of HA did not inuence cell viability in our study. 3-D bioprinting offers the possibility of printing hollow channels to provide nutrients to cells, and was shown to enhance cell viability of broblasts encapsulated in a 3-D bioprinted collagen hydrogel [25]. This method is enhanced further by printing two distinct phases next to each other as demonstrated in a proof-ofconcept study, where we printed tubular structures within a bulk phase as shown in Fig. 8 to demonstrate a simple approach towards engineering of larger 3-D tissues. Channel structures and two-phase constructs exhibit signicant potential to be enlarged and may eventually be used as whole vascularized systems, which would be an important step forward in tissue engineering. Potential applications for the system presented here are manifold. We used the addition of HA to the bioink as one example to show the versatility of the system. Tissue engineering of bone requires a matrix which stimulates osteogenic differentiation and mineralization. Hydroxyapatite was chosen due to its potential osteogenic promoter characteristics [18]. For the application of this process for other tissues such as cartilage, kidney, liver, muscles or vasculature, it needs to be investigated whether enhancing factors other than HA can be integrated and how they interact with this system. Moreover, as the process itself guaranteed cell survival, it seems possible that sensitive drugs of drug delivery systems could be incorporated without losing their bioactivity. The printing approach offers the possibility to place cells in a predened location in a 3-D matrix. The different materials can be combined in multiphase printed constructs. Another possible application would be the engineering of osteochondral tissue, for example, osteochondral plugs. With conventional seeding methods only one cell type can be seeded at the same time and thus it is up to the cells to produce bone tissue on one side and cartilage on the other, which is very challenging to achieve. Other approaches where cartilage and bone tissue are cultured separately and merged before implantation typically resulted in weak osteochondral interfaces. Using 3D bioprinting to generate osteochondral tissue can help to overcome these limitations. Both cell types can be placed next to each other within the same construct and develop not only cartilage and bone tissue, but also a strong interface in vitro. The advantage of using this versatile hydrogel composite to engineer multi-tissue constructs is the similarity of the swelling properties of the hydrogels with and without HA such that no phase separation due to swelling takes place.

5. Conclusion A hydrogel composite based on an alginategelatin hydrogel precursor was successfully modied with different amounts of

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HA for 3-D bioprinting in combination with a two-step crosslinking procedure to generate stable 3-D bioprinted constructs. The hardware of the 3-D bioprinter was reconstructed to address the temperature-dependent gelation of gelatin as an instantaneous stabilizer. Improved temperature control and a more homogeneous heat distribution in the bioink were achieved by the newly developed syringe tip heaters. Living cells could be included in the bioink and showed a high viability after the printing and crosslinking processes. Augmentation with HA led to enhanced visibility of the hydrogel in lCT and an increased gel point with a slight loss in lament homogeneity. Swelling properties as well as mechanical stiffness were kept in a similar range. We could show that printability of the constructs was maintained by adding HA in different concentrations to the alginategelatin hydrogel. Even with the increased shear stress during printing, which results from higher amounts of HA, no reduction of cell viability of encapsulated cells 3 days after printing was observed. We successfully printed singleand multi-layer objects including cells and a two-phase construct consisting of repeated tubes. The geometry of the printed samples remained stable thanks to the combined two-step crosslinking process. It can be concluded that the presented materials and processes provide a highly versatile system for further 3-D bioprinting studies. Acknowledgments The presented work was funded by the RMS Foundation (Bettlach, Switzerland). This work was also supported by a scholarship from the Whitaker International Program. The Whitaker International Program is administered by the Institute of International Education (IIE). We acknowledge Roman Schneider from the Institute for Biomechanics, ETH Zurich, for his help in hydrogel development and characterization, Dr. Marc Bohner, Dr. Nicola Dbelin and Latitia Galea from RMS Foundation for the characterization of HA powder and Dr. Marc A. Gauthier from the Institute of Pharmaceutical Sciences, ETH Zurich, for his support concerning rheology. Technical advice and hardware modication of the 3-D bioprinter was provided by Peter Schwilch and Marco Hitz from the Institute for Biomechanics, ETH Zurich. Appendix A. Figures with essential colour discrimination Certain gures in this article, particularly Figs. 1 and 7 are difcult to interpret in black and white. The full colour images can be found in the on-line version, at http://dx.doi.org/10.1016/ j.actbio.2013.10.016. Appendix B. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2013. 10.016.

References
[1] Langer R, Vacanti J. Tissue engineering. Science 1993;260:9206. [2] Thimm BW, Wst S, Hofmann S, Hagenmller H, Mller R. Initial cell precultivation can maximize ECM mineralization by human mesenchymal stem cells on silk broin scaffolds. Acta Biomater 2011;7:221828. [3] Uebersax L et al. Effect of scaffold design on bone morphology in vitro. Tissue Eng 2006;12:341729. [4] Nazarov R, Jin H-J, Kaplan DL. Porous 3-D scaffolds from regenerated silk broin. Biomacromolecules 2004;5:71826. [5] Wst S, Mller R, Hofmann S. Controlled positioning of cells in biomaterialsapproaches towards 3D tissue printing. J Funct Biomater 2011;2:11954. [6] Landers R, Pster A, Hbner U, John H, Schmelzeisen R, Mlhaupt R. Fabrication of soft tissue engineering scaffolds by means of rapid prototyping techniques. J Mater Sci 2002;37:310716. [7] Tian XY, Li MG, Cao N, Li JW, Chen XB. Characterization of the ow behavior of alginate/hydroxyapatite mixtures for tissue scaffold fabrication. Biofabrication 2009;1:045005. [8] Derby B. Printing and prototyping of tissues and scaffolds. Science 2012;338:9216. [9] Fedorovich NE, Wijnberg HM, Dhert WJA, Alblas J. Distinct tissue formation by heterogeneous printing of osteo- and endothelial progenitor cells. Tissue Eng Part A 2011;17:211321. [10] Xu M, Wang X, Yan Y, Yao R, Ge Y. An cell-assembly derived physiological 3D model of the metabolic syndrome, based on adipose-derived stromal cells and a gelatin/alginate/brinogen matrix. Biomaterials 2010;31:386877. [11] Yan Y et al. Fabrication of viable tissue-engineered constructs with 3D cellassembly technique. Biomaterials 2005;26:586471. [12] DEste M, Eglin D. Hydrogels in calcium phosphate moldable and injectable bone substitutes: sticky excipients or advanced 3-D carriers? Acta Biomater 2013;9:542130. [13] Fedorovich NE, Alblas J, de Wijn JR, Hennink WE, Verbout AJ, Dhert WJ. Hydrogels as extracellular matrices for skeletal tissue engineering: state-ofthe-art and novel application in organ printing. Tissue eng 2007;13: 190525. [14] Nicodemus GD, Bryant SJ. Cell encapsulation in biodegradable hydrogels for tissue engineering applications. Tissue Eng Part B 2008;14:14965. [15] Mironov V, Prestwich G, Forgacs G. Bioprinting living structures. J Mater Chem 2007;17:205460. [16] Tadic D, Epple M. A thorough physicochemical characterisation of 14 calcium phosphate-based bone substitution materials in comparison to natural bone. Biomaterials 2004;25:98794. [17] Lin H-R, Yeh Y-J. Porous alginate/hydroxyapatite composite scaffolds for bone tissue engineering: preparation, characterization, and in vitro studies. J Biomed Mater Res B Appl Biomater 2004;71B:5265. [18] Fedorovich NE et al. Biofabrication of osteochondral tissue equivalents by printing topologically dened, cell-laden hydrogel scaffolds. Tissue Eng Part C 2012;18:3344. [19] Hofmann S et al. Control of in vitro tissue-engineered bone-like structures using human mesenchymal stem cells and porous silk scaffolds. Biomaterials 2007;28:115262. [20] Cui X, Boland T. Human microvasculature fabrication using thermal inkjet printing technology. Biomaterials 2009;30:62217. [21] Fedorovich NE, De Wijn JR, Verbout AJ, Alblas J, Dhert WJ. Three-dimensional ber deposition of cell-laden, viable, patterned constructs for bone tissue printing. Tissue Eng Part A 2008;14:12733. [22] Whitesides GM, Grzybowski B. Self-assembly at all scales. Science 2002;295:241821. [23] Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specication. Cell 2006;126:67789. [24] Pittenger MF et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:1437. [25] Lee W et al. On-demand three-dimensional freeform fabrication of multilayered hydrogel scaffold with uidic channels. Biotechnol Bioeng 2010;105:117886.