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Hybrid hierarchical fabrication of three-dimensional scaffolds
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Technical Paper
Hybrid hierarchical fabrication of three-dimensional scaffolds Chuang Wei b , Jingyan Dong a,∗ a Edward P. Fitts Department of Industrial and Systems Engineering, North Carolina State University, 414-C Daniels Hall, Campus box 7906, Raleigh, NC 27695-7906, USA b Edward P. Fitts Department of Industrial and Systems Engineering, North Carolina State University, 425 Daniels Hall, Campus box 7906, Raleigh, NC 27695-7906, USA
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Article history: Received 6 May 2013 Accepted 10 October 2013 Available online xxx Keywords: Tissue engineering Hierarchical scaffolds Free-form fabrication Electrohydrodynamic melt jet plotting
a b s t r a c t Three-dimensional (3D) porous structures facilitating cell attachment, growth, and proliferation is critical to tissue engineering applications. Traditional solid freeform fabrication (SFF) methods have limited capabilities in the fabrication of high resolution micro-scale features to implement advanced biomedical functions. In this work, we present a hybrid scaffold fabrication approach by integrating electrohydrodynamic (EHD) printing technology with extrusion deposition together to fabricate hierarchical 3D scaffolds with well controlled structures at both macro and micro scale. We developed a hybrid fabrication platform and a robust fabrication process to achieve 3D hierarchical structures. The melting extrusion by pneumatic pressure was used to fabricate 3D scaffolds with filaments dimension of hundreds of microns using thermoplastic biopolymer polycaprolactone (PCL). An electrohydrodynamic (EHD) melt jet plotting process was developed to fabricate micro-scale features on the scaffolds with sub-10 m resolution, which has great potential in advanced biomedical applications, such as cell alignment and cell guidance. © 2013 The Society of Manufacturing Engineers. Published by Elsevier Ltd. All rights reserved.
1. Introduction Tissue engineering scaffolds for tissue regeneration have many specific requirements on materials and structural features that demand different fabrication capabilities. Even though the detailed requirement varies with applications, the general requirements [1] include the biocompatibility and biodegradability of the material, pore size and pore distribution that determine the mass-transport property, and the surface properties that determine cell adhesion and cell alignment. While the relatively large structures and pores (generally >100 m) are important for providing mechanical support and material transportation, micro-scale structures with their dimension similar to the size of the cell provide many advanced capabilities to regulate cell responses to the scaffold [2–3], such as cell alignment and cell guidance. Conventional fabrication technologies [4–8], such as solvent casting, phase separation, gas forming, freeze-drying particulateleaching, lack capabilities to precisely control the structural features such as porosity and pore size of the resulting scaffolds. Solid freeform fabrication (SFF) technologies [9] (e.g. stereolithography [10], selective laser sintering [11], 3D printing (3DP) [12], and fused deposition modeling (FDM) [13]) are widely used in scaffold fabrication, which can precisely control the geometric feature of the scaffolds with a broad range of materials, including
∗ Corresponding author. Tel.: +1 919 515 7196; fax: +1 919 515 5281. E-mail addresses: [email protected] (C. Wei), [email protected] (J. Dong).
photo-crosslinkable bio-polymers, thermoplastic polymers, ceramics or composite materials. Widely used extrusion and ink-jet printing based solid freeform fabrication methods are limited in their achievable structural resolution that is mainly controlled by the nozzle size. FDM [13] and precision extrusion deposition (PED) [14,15] are capable of layered fabrication of 3D structures for thermoplastic biodegradable polymers, such as polycaprolactone (PCL). However, scaling down the nozzle size for better resolution will make the required extrusion pressure unpractical high for high viscous biopolymers, since the extrusion pressure scales up much faster when the nozzle diameter is decreased according to Hagen–Poiseuille equation. Electrohydrodynamic (EHD) printing is a high resolution printing method, in which the printed materials, ranging from polymer solution or polymer melt, ceramic solution to composite materials, are subjected to high electrostatic field to form a Taylor-cone structure and a fine jet that ejects from the cone. The diameter of the jet is significantly smaller than the nozzle diameter, which can overcome the limitation of the nozzle size and produce micro and nano-scale features. Depending on the parameters used in EHD printing, EHD behavior can be categorized into different operational modes [16], including electrospray [17], electrospinning [18], and electro-hydrodynamic jet printing [19]. These parameters include material properties of the ink (e.g., viscosity, density, conductivity, and permeability) and operational conditions such as applied voltage/electrical field, pressure/flow rate, and substrate to nozzle distance. Electrospray utilizes the unstable electrohydrodynamic behavior to form extremely small while uniform droplet.
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Please cite this article in press as: Wei C, Dong J. Hybrid hierarchical fabrication of three-dimensional scaffolds. J Manuf Process (2013), http://dx.doi.org/10.1016/j.jmapro.2013.10.003
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Electrospinning can produce micro-scale or nano-scale continuous fibers by ejecting a polymer jet from the Taylor cone when the electric field strength exceeds the surface tension of the liquid. The fiber jet travels through a long distance experiencing whipping instability in most cases to evaporate the solvent. Since electrospray and electrospinning take the advantage of the instability regime of electrohydrodynamic cone-jet, it is difficult to be directly applied to the controlled fabrication of high precision micro-structures. Working in a stable jet region is critically important for the freeform fabrication of three dimensional structures using electrohydrodynamic jet printing. Depending on different process conditions, axis-symmetric instability, in which droplets break-up from the jet, or nonaxis-symmetric instability (i.e. whipping) will disrupt the stable cone jet [20,21]. These instabilities are required for electrospray and electrospinning, but devastating for precision 2D and 3D fabrication. Process parameters as well as materials used have to be carefully chosen to obtain a stable cone-jet. For some extreme cases [22], very low conductivity liquids such as toluene and hexane may not have any cone-jet mode. There exists a minimum flow rate threshold for cone-jet mode [23], and a maximum flow rate [24] above which whipping instability is developed. For the fabrication of three-dimensional structure, the upper layer filaments need to span over the gap underneath them. Quick solidification of the printed filaments against sagging down effect is also critical. In this study, we present a hybrid scaffold fabrication approach by integrating electrohydrodynamic (EHD) printing technology with extrusion deposition approach together to fabricate 3D scaffolds with well controlled structures at both the macro scale and micro scale. Polycaprolactone (PCL) is used in this study. The hierarchical 3D structures include thick filaments with the diameter of hundreds of microns and thin filaments with sub-10 m dimensions. The thick filaments are fabricated by the melting extrusion layer-by-layer as the backbone to provide mechanical support. After each thick layer is deposited, small filaments with sub-10 m resolution are deposited by electrohydrodynamic (EHD) hot jet plotting process to fabricate micro-scale features on the scaffolds. After 3D scaffold fabrication, the morphologies and microstructures have been characterized by scanning electron microstructures (SEM). Besides the traditional functions of the scaffolds, the hierarchical scaffold structures can provide additional potentials in advanced biomedical applications, such as cell alignment and guidance [25,26].
and melting point from 56 to 64 ◦ C. As a biocompatible and biodegradable polymer that has been approved by the FDA (Food and Drug Administration), polycaprolactone has been widely used for the fabrication of tissue engineering scaffolds. The favorable rheological and thermo stability also make PCL a great candidate to be used in the fabrication of micro-scale structures by EHD-jet plotting in its melting phase. The temperature gradient can solidify the jetted PCL to form well controlled fine filaments, which is highly preferable for the fabrication of 3D structures. 2.2. System setup for hierarchical scaffold fabrication The high-precision hierarchical fabrication platform (Fig. 1) was composed of four sub-systems: a three-axis (XYZ) precision stage, a pneumatic dispensing unit, an EHD hot jet plotting unit, and a thermal control system. The XYZ precision stage was located on an optical table to reduce vibrational noise on which three linear stages were configured in XYZ directions with 100 nm repeatability and a displacement range of 100 × 100 × 50 mm. A high resolution camera with a maximum resolution of 0.5 m was used to monitor the deposition process. There are two deposition units for deposit PCL structures, one for pneumatic thermal extrusion, and one for EHD hot jet plotting. The temperature for both thermal extrusion and EHD hot jet plotting is kept at 82 ◦ C degree to obtain the favorable rheology of the melting polymer. The nozzle for pneumatic thermal extrusion has a 250 m ID and 450 m OD. The nozzle for EHD jet plotting has a 150 m ID and 300 m OD. The pneumatic system can provide a maximum pressure of 80 psi (550 kPa). The electrical potential between the nozzle and the substrate in EHD plotting is controlled by a high voltage source meter (TREK 610E) that can supply up to 10 K V. The temperatures of heated syringes are monitored by thermo-couple and closed-loop controlled by a temperature controller with 0.5 ◦ C degree resolution. The substrate is the glass slide rests on an aluminum coated silicon wafer that provides an electrically grounded support. The substrate and the underneath ground electrode was displaced by the XY-stage. The G-code program is used to generate the tool path of the printing system in this study. In case of complex pattern, computer-aided design (CAD) software package can be used to generate the G-code for a specific structure design then feed into the motion controller to provide the motion of the printing system. 2.3. EHD dispensing process characterization
2. Materials and methods 2.1. Materials for hybrid fabrication Polycaprolactone (PCL) pellets were purchased from Sigma–Aldrich (Milwaukee, WI) with average Mn of 45,000 g/mol
In EHD jet printing, a high voltage is applied to the nozzle tip, which causes mobile ions in the printed material (melted PCL in this study) to accumulate on the PCL/air interface. The Coulombic force causes the meniscus at the nozzle tip to deform into a conical shape (i.e., Taylor cone). With sufficiently large electric field, the
Fig. 1. Schematic of the hybrid fabrication system for E-jet printing and thermal extrusion deposition (a) EHD plotting unit. (b) Pneumatic extrusion unit. (c) The experimental testbed.
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Back Pressure Nozzle Surface Tension
Electrostatic stress
Viscous Force Whipping Region Fig. 2. Forces on Taylor cone in EHD processing.
surface charge repulsion from the electrostatic stress at the cone apex exceeds the surface tension of the meniscus, and a droplet or a jet of fluid is printed from the Taylor cone onto the grounded substrate (Fig. 2). The fluid properties of the printed material, along with the process conditions, mostly applied voltage, pressure, printing speed, and nozzle–substrate distance, determines the printing results. In this study, short nozzle–substrate distance (250 m) was chosen to utilize the stable jet region and avoid potential whipping instability. The effects of other process parameters (applied voltage, pressure, and printing speed) on the melt EHD-jet plotting process was investigated to obtain the most appropriate set of process conditions for 3D structure fabrication. 2.4. Hierarchical scaffold fabrication process The fabrication process of PCL hierarchical structures follows a layer-by-layer fashion as in Fig. 3. Step 1: A thick layer of PCL filaments with centerline space 500 m is deposited by pneumatic thermal extrusion following the programmed tool path. The process condition at this step uses the pressure of 60 psi, feedrate of 1.0 mm/s, and the nozzle-to-substrate distance of 250 m. Step 2: Micro-filament layers are dispensed by EHD hot jet plotting process onto the thick PCL structures. The orientation of
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micro-filament and thick PCL filaments can be selected by changing the plotted tool path. In this study, two orientations of the micro filaments are applied. One is along and perpendicular to the underneath thick filaments with 75 m centerline spacing. The other one is 45 and 135 degree to the underneath thick filaments with 85um spacing. This step deposits a well-connected micro filament network on the backbone thick filament structure. During EHD plotting, the pressure is chosen to be 4 psi, the feed rate is 3.0–5.0 mm/s, and the standoff height is 250 m, the applied voltage for each layer is from 1600 V to 2000 V. Step 3: Changing the orientation of the deposited structures, and repeat Step 1 and Step 2 until the desired thickness is achieved. 2.5. SEM morphology characterization The shape of the cone-jet of the electrohydrodynamic jet printing was observed by the high resolution video camera. The morphology of hierarchical scaffold structures was characterized by SEM (S-3500, Hitachi Instruments Inc., Tokyo, Japan). The scaffolds were sputter-coated with gold, and viewed at 5 kV accelerating voltage. The filament size and spacing were measured and calculated from SEM images using the image analysis software. 3. Results and discussion 3.1. Characterization of EHD hot jet plotting process In EHD printing, the applied electrical field deforms the meniscus at the nozzle tip into the Taylor cone, and eventually produces a jet from the cone shape. Generally, as the applied electric field strength increases, different electrohydrodynamic printing modes can be observed, transitioning from pulsating mode to stable jet, and then to unstable multiple jets [16]. For the melted polycaprolactone, when the nozzle to substrate gap was fixed, as we increased the voltage from 500 V, a meniscus and Taylor cone was gradually forming at the tip of the nozzle (Fig. 2a). However at a voltage lower than 800 V, there is no observed drip and jet generated from the cone. When the voltage was increased to about 825 V, a stable jet began to be ejected from the cone. As voltage increased, the stable jet remains, but both the radius and elongation of the cone became smaller (Fig. 4b and c). When the applied voltage was increased to about 1100 V unstable multi-jets appears, as shown in Fig. 4d. In our experimental condition, we did not observe any pulsating printing mode (single drop printed onto the substrate), partially due to
Fig. 3. The fabrication process of the hierarchical structures with different orientations between thick filaments and micro-filaments. (a) and (a1) Deposition of thick backbone filaments. (b) and (b1) Deposition of micro filaments network. (c) and (c1) Deposition another backbone filaments layer. (d) and (d1) Deposition of another layer of micro filaments network.
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Fig. 4. (a) Small meniscus without jet or drip at nozzle tip at 600 V. (b) and (c) Stable cone-jet at 900 V and 1050 V. (d) Unstable multi-jet at high electric field strength.
the high viscosity of the melted polycaprolactone. It is difficult for melted PCL to obtain enough electric stress to overcome the surface tension at the meniscus to form a fine drop. Obtaining stable electrohydrodynamic cone-jet mode requires the proper selection of the process parameters that include electrical voltage, pressure and plotting speed. These process conditions need to be thoroughly investigated to achieve reliable jet printing and filament formation. In this study, small nozzle–substrate gap was chosen to achieve large enough electric strength to form the stable cone-jet. At the small standoff height, the jet is kept at the stable and straight region, and the effect of whipping instability is minimized. To observe the effect of different process conditions on the cone-jet shape and the plotted filaments, one process parameter (from electrical voltage, pressure and plotting speed) was changed at a time while keeping others constant. The applied voltage played the most important role in the EHDjet printing process. Clearly there exists a lower bound threshold voltage (between 800 V and 825 V for this configuration) to continuously jet melted polymer. At a relative low voltage, a skewed cone-jet was observed as shown in Fig. 5(a1), in which the jet was inclined to the substrate along the plotting direction. As voltage increased, the skew cone-jet was changed to straight cone-jet. After the voltage was further increased, although a straight and stable
cone-jet was observed from the camera, the plotted filaments became wavy and lose controllability for precision fabrication. The amplitude of waviness increased with the increase of the voltage. This behavior can be explained by the specific material property of PCL and the jetting speed. In EHD printing, a larger voltage indicates a larger electrostatic stress, and then larger acceleration and jetting speed. When plotting speed is fixed, at the low voltage, the jetting speed was lower than the plotting speed of the stage, the high viscous force of the melted PCL deformed the cone-jet to give the skewed cone-jet. When the jetting speed matched the plotting speed, a straight cone-jet was obtained. When the jetting speed exceeds the plotting speed at high voltage, the high viscosity of the plotted and semi-solidified PCL filaments makes it difficult to reflow on the substrate. Thus the wavy filaments were obtained to absorb the over-plotted length due to speed mismatch. It is very important to select the plotting speed to match the jetting speed. The mismatch between the jetting speed and the plotting speed will significantly affect the cone-jet shape and the printed filaments. If other process parameters (voltage and pressure) are selected, the jet ejecting speed from the cone is fixed. When plotting at a small speed, wavy filaments were observed (Fig. 5(b1)). As the plotting speed increased, the filaments become straight gradually. After the plotting speed was further increased,
Fig. 5. The cone-jet shape and jet printed filaments at different conditions. (a1)–(a3) Different voltage, (b1)–(b3) different feedrate, (c1)–(c3) different pressure.
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the skewed cone-jet appeared with straight filaments left on the substrate. This specific behavior indicated the importance of matching the plotting speed with the jetting speed. For the straight filaments fabricated by the stable cone-jet and slightly skewed cone-jet, the plotting speed significantly changed the plotted filament width. The line width decreased from about 25 m to 17 m, as plotting speed increased from 1.2 mm/s to 2.4 mm/s. The results indicate that the PCL jet or semi-solidified filament can be stretched with reduced filament diameter. The plotting speed can be used as a method to adjust of the dimension of the fabricated features. It is well known that the pressure or the flow rate can affect cone-jet shape and jet stability [22]. The required pressure range for maintaining stable-jet plotting is determined by the nozzle size and other process conditions. In EHD printing, generally larger flow rate will make the jet less stable and produce whipping instability. However, for EHD-jet printing of melted PCL, we observed vibrating jet and wavy filament at the low pressure (Fig. 5(c1)). As pressure increased, the stable jet and the skewed cone-jet appeared gradually, and straight filaments were achieved. This phenomenon is again the result of the high viscosity of the melted PCL jet and interaction between the cone-jet and the printed filaments on the substrate. With a small flow rate at low pressure, a thin jet was obtained. The electrostatic stress provided large acceleration and high speed of the ejected jet. This jetting speed was larger than the plotting speed, thus wavy filaments and vibrating jet were observed. As pressure increased, large flow rate produced a thick jet. With similar electric stress (from the same voltage), less acceleration was applied to the jet, resulting smaller jetting speed. Thus the jetting speed gradually matched the plotting speed, and then was lower than the plotting speed. As a result, straight filaments and skewed jet were observed. This explanation was further supported by the increased filament width (indicating larger flow rate) along with the increase of the pressure.
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3.2. Hierarchical structure fabrication PCL is an excellent biomaterial for thermal extrusion due to its thermal stability and quick solidification. The large viscosity of melted PCL enables its direct extrusion without any supporting material. However, the large viscosity leads to the difficulty in the direct deposition of high resolution features due to the high pressure requirement. Even though higher melting temperature can reasonably lower the viscosity, the thermal stress during the solidification of melting polymer limits the usable range of the melting temperature. In the study, for the thermal extrusion of thick filaments, 180 ◦ F or 82 ◦ C is used with the pressure, feedrate and layer thickness maintained constant during the whole process at 60 psi, 1.0 mm/s and 250 m respectively. High resolution (sub-10 m) fabrication of 2D patterns and 3D structures were achieved using the EHD-jet plotting of melted polycaprolactone (Fig. 6). The smallest line width was about 5 m, which are at least one to two orders of magnitude smaller than direct melt extrusion based fabrication approaches. Because a small nozzle–substrate gap, there is less lagged jet/filament in-between the nozzle and the substrate. As a result, the EHD-jet plotting process can respond well to sharp geometric change. Fig. 6(c) and (d) demonstrates a porous 3D lattice-structured scaffold of PCL with fully interconnected inner architectures that were fabricated using the EHD hot jet plotting approach. The uniformity of the pores and the filaments indicated the applicability of using the developed EHD hot jet plotting process to fabricate structures at the micro-scale. Fabrication of hierarchical 3D scaffold structures with both large and micro filaments is very challenging, because the microfilaments need to span over a large gap (more than 250 m) without any support. Moreover, the structures deposited on the substrate essentially change the electrode geometry and charge distribution, which brings very complex perturbation to the electrostatic field
Fig. 6. High resolution two-dimensional patterns (a) and (b) and three-dimensional scaffold structure (c) and (d) fabricated by direct EHD-jet plotting of melted PCL.
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is too low, besides the winding filaments as mentioned previously, filament sagging down becomes significant, since the micro filament needs to span over more than 250 m gap. Without enough plotting speed, the jetted filaments will have severe sag down. If the plotting velocity is too high, the filaments have a large percentage for breaking up due to the thermal stress and weak mechanical structure. The effect of deposited structures on the electrostatic field strength also significantly affects the plotting performance, especially when there are an orientation angle between macrofilaments and micro-filaments. The deposited structures are acting as electrodes and will accelerate the jet around them. The jet velocity in the free space is much lower than the vicinity around the thick filaments, which is the reason why the angle on large filaments is steeper while the angle over space is smaller. It is very complicated to tune the highly coupled processing parameters. The EHD parameters for each single layer are different because the printed structures are essentially changing the collector geometry and perturb the electrostatic field and distribution. However, once these parameters are well-adjusted, the process is robust enough to obtain well-connected micro filaments network with good quality and repeatability In future, a detailed EHD plotting model will be developed to understand the EHD process and help to optimize the process parameters. 4. Conclusion
Fig. 7. SEM images of the hierarchical 3D scaffold structures.
strength and distribution, and makes the parameter tuning very difficult. During the hierarchical fabrication, we kept the standoff height between the tip and feature top surface while adjusting the applied voltage to count for the change of electrostatic field strength due to the plotted layers on the substrate. The major problem of micro filaments fabrication is their breakup when spanning over the large gap between two thick filaments. The process conditions need to be thoroughly investigated to achieve reliable jet printing and reliable filament formation. We observed that filament formation performance can be improved by decreasing feed rate and increasing the pressure, which can effectively increase micro-filament dimension for better mechanical strength. The fabricated hierarchical 3D scaffolds are shown in Fig. 7. In this study, we have fabricated two groups of scaffolds having the same macro-structure but microstructure with different orientation. The porous hierarchical 3D structures have fully interconnected inner architectures, and micro filaments are fabricated using the EHD jet plotting technology. The uniformity of pore size and the micro filaments network demonstrate the capabilities of the hybrid process for the fabrication of hierarchical scaffold structures. The hierarchical PCL scaffolds have a macro pore size of 230 ± 10 m and a porosity of 50%, with sub-10 m micro filaments, which provides the required pore size and mechanical support to allow for cell proliferation, migration, and ingrowth into the scaffolds. Particularly, the micro features can be customized to tailor the interaction between the cell and the scaffold, and provide advanced biomedical functions, such as cell alignments and cell guidance. In the hierarchical fabrication of 3D structures, choosing a proper EHD plotting speed is a critical step. When the plotting speed
In the paper, a hybrid scaffold fabrication approach is presented, which integrates electrohydrodynamic (EHD) printing technology with extrusion deposition together to fabricate 3D hierarchical scaffolds with well controlled structures at both the macro scale and micro scale. In this study, a hybrid fabrication platform and a robust fabrication process to achieve 3D hierarchical structures are developed. The melting extrusion by pneumatic pressure is used to fabricate 3D scaffolds using thermoplastic biopolymer polycaprolactone (PCL) with filaments dimension of hundreds of microns. An electrohydrodynamic (EHD) hot jet plotting process is developed to fabricate micro-scale features on the scaffolds with sub-10 m resolution, which has great potential in advanced biomedical applications, such as cell alignment and guidance. Acknowledgements The authors gratefully acknowledge support from the National Science Foundation under Grant Award CMMI 1129817 and CMMI1333775. References [1] Yang S, Leong KF, Du Z, Chua CK. The design of scaffolds for use in tissue engineering. Part I. Traditional factors. Tissue Eng 2010;7(6):679–89. [2] Sachlos E, Czernuszka JT. Making tissue engineering scaffolds work. Review: the application of solid freeform fabrication technology to the production of tissue engineering scaffolds. Eur Cell Mater 2003;5(29):39–40. [3] Tay CY, Irvine SA, Boey FY, Tan LP, Venkatraman S. Micro-/nano-engineered cellular responses for soft tissue engineering and biomedical applications. Small 2011;7:1361–78. [4] Misra SK, Ansari TI, Valappil SP, Mohn D, Philip SE, Stark WJ, et al. Poly(3hydroxybutyrate) multifunctional composite scaffolds for tissue engineering applications. Biomaterials 2010;31:2806–15. [5] Rowlands AS, Lim SA, Martin D, Cooper-White JJ. Polyurethane/poly(lacticco-glycolic) acid composite scaffolds fabricated by thermally induced phase separation. Biomaterials 2007;28:2109–21. [6] Chen W, Zhou H, Tang M, Weir MD, Bao C, Xu HHK. Gas-foaming calcium phosphate cement scaffold encapsulating human umbilical cord stem cells. Tissue Eng A 2011;18:816–27. [7] Caliari SR, Ramirez MA, Harley BA. The development of collagen-GAG scaffold-membrane composites for tendon tissue engineering. Biomaterials 2011;32(34):8990–8. [8] Hutmacher DW. Scaffolds in tissue engineering bone and cartilage. Biomaterials 2000;21(24):2529–43.
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Technical Paper
Hybrid hierarchical fabrication of three-dimensional scaffolds Chuang Wei b , Jingyan Dong a,∗ a Edward P. Fitts Department of Industrial and Systems Engineering, North Carolina State University, 414-C Daniels Hall, Campus box 7906, Raleigh, NC 27695-7906, USA b Edward P. Fitts Department of Industrial and Systems Engineering, North Carolina State University, 425 Daniels Hall, Campus box 7906, Raleigh, NC 27695-7906, USA
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Article history: Received 6 May 2013 Accepted 10 October 2013 Available online xxx Keywords: Tissue engineering Hierarchical scaffolds Free-form fabrication Electrohydrodynamic melt jet plotting
a b s t r a c t Three-dimensional (3D) porous structures facilitating cell attachment, growth, and proliferation is critical to tissue engineering applications. Traditional solid freeform fabrication (SFF) methods have limited capabilities in the fabrication of high resolution micro-scale features to implement advanced biomedical functions. In this work, we present a hybrid scaffold fabrication approach by integrating electrohydrodynamic (EHD) printing technology with extrusion deposition together to fabricate hierarchical 3D scaffolds with well controlled structures at both macro and micro scale. We developed a hybrid fabrication platform and a robust fabrication process to achieve 3D hierarchical structures. The melting extrusion by pneumatic pressure was used to fabricate 3D scaffolds with filaments dimension of hundreds of microns using thermoplastic biopolymer polycaprolactone (PCL). An electrohydrodynamic (EHD) melt jet plotting process was developed to fabricate micro-scale features on the scaffolds with sub-10 m resolution, which has great potential in advanced biomedical applications, such as cell alignment and cell guidance. © 2013 The Society of Manufacturing Engineers. Published by Elsevier Ltd. All rights reserved.
1. Introduction Tissue engineering scaffolds for tissue regeneration have many specific requirements on materials and structural features that demand different fabrication capabilities. Even though the detailed requirement varies with applications, the general requirements [1] include the biocompatibility and biodegradability of the material, pore size and pore distribution that determine the mass-transport property, and the surface properties that determine cell adhesion and cell alignment. While the relatively large structures and pores (generally >100 m) are important for providing mechanical support and material transportation, micro-scale structures with their dimension similar to the size of the cell provide many advanced capabilities to regulate cell responses to the scaffold [2–3], such as cell alignment and cell guidance. Conventional fabrication technologies [4–8], such as solvent casting, phase separation, gas forming, freeze-drying particulateleaching, lack capabilities to precisely control the structural features such as porosity and pore size of the resulting scaffolds. Solid freeform fabrication (SFF) technologies [9] (e.g. stereolithography [10], selective laser sintering [11], 3D printing (3DP) [12], and fused deposition modeling (FDM) [13]) are widely used in scaffold fabrication, which can precisely control the geometric feature of the scaffolds with a broad range of materials, including
∗ Corresponding author. Tel.: +1 919 515 7196; fax: +1 919 515 5281. E-mail addresses: [email protected] (C. Wei), [email protected] (J. Dong).
photo-crosslinkable bio-polymers, thermoplastic polymers, ceramics or composite materials. Widely used extrusion and ink-jet printing based solid freeform fabrication methods are limited in their achievable structural resolution that is mainly controlled by the nozzle size. FDM [13] and precision extrusion deposition (PED) [14,15] are capable of layered fabrication of 3D structures for thermoplastic biodegradable polymers, such as polycaprolactone (PCL). However, scaling down the nozzle size for better resolution will make the required extrusion pressure unpractical high for high viscous biopolymers, since the extrusion pressure scales up much faster when the nozzle diameter is decreased according to Hagen–Poiseuille equation. Electrohydrodynamic (EHD) printing is a high resolution printing method, in which the printed materials, ranging from polymer solution or polymer melt, ceramic solution to composite materials, are subjected to high electrostatic field to form a Taylor-cone structure and a fine jet that ejects from the cone. The diameter of the jet is significantly smaller than the nozzle diameter, which can overcome the limitation of the nozzle size and produce micro and nano-scale features. Depending on the parameters used in EHD printing, EHD behavior can be categorized into different operational modes [16], including electrospray [17], electrospinning [18], and electro-hydrodynamic jet printing [19]. These parameters include material properties of the ink (e.g., viscosity, density, conductivity, and permeability) and operational conditions such as applied voltage/electrical field, pressure/flow rate, and substrate to nozzle distance. Electrospray utilizes the unstable electrohydrodynamic behavior to form extremely small while uniform droplet.
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Electrospinning can produce micro-scale or nano-scale continuous fibers by ejecting a polymer jet from the Taylor cone when the electric field strength exceeds the surface tension of the liquid. The fiber jet travels through a long distance experiencing whipping instability in most cases to evaporate the solvent. Since electrospray and electrospinning take the advantage of the instability regime of electrohydrodynamic cone-jet, it is difficult to be directly applied to the controlled fabrication of high precision micro-structures. Working in a stable jet region is critically important for the freeform fabrication of three dimensional structures using electrohydrodynamic jet printing. Depending on different process conditions, axis-symmetric instability, in which droplets break-up from the jet, or nonaxis-symmetric instability (i.e. whipping) will disrupt the stable cone jet [20,21]. These instabilities are required for electrospray and electrospinning, but devastating for precision 2D and 3D fabrication. Process parameters as well as materials used have to be carefully chosen to obtain a stable cone-jet. For some extreme cases [22], very low conductivity liquids such as toluene and hexane may not have any cone-jet mode. There exists a minimum flow rate threshold for cone-jet mode [23], and a maximum flow rate [24] above which whipping instability is developed. For the fabrication of three-dimensional structure, the upper layer filaments need to span over the gap underneath them. Quick solidification of the printed filaments against sagging down effect is also critical. In this study, we present a hybrid scaffold fabrication approach by integrating electrohydrodynamic (EHD) printing technology with extrusion deposition approach together to fabricate 3D scaffolds with well controlled structures at both the macro scale and micro scale. Polycaprolactone (PCL) is used in this study. The hierarchical 3D structures include thick filaments with the diameter of hundreds of microns and thin filaments with sub-10 m dimensions. The thick filaments are fabricated by the melting extrusion layer-by-layer as the backbone to provide mechanical support. After each thick layer is deposited, small filaments with sub-10 m resolution are deposited by electrohydrodynamic (EHD) hot jet plotting process to fabricate micro-scale features on the scaffolds. After 3D scaffold fabrication, the morphologies and microstructures have been characterized by scanning electron microstructures (SEM). Besides the traditional functions of the scaffolds, the hierarchical scaffold structures can provide additional potentials in advanced biomedical applications, such as cell alignment and guidance [25,26].
and melting point from 56 to 64 ◦ C. As a biocompatible and biodegradable polymer that has been approved by the FDA (Food and Drug Administration), polycaprolactone has been widely used for the fabrication of tissue engineering scaffolds. The favorable rheological and thermo stability also make PCL a great candidate to be used in the fabrication of micro-scale structures by EHD-jet plotting in its melting phase. The temperature gradient can solidify the jetted PCL to form well controlled fine filaments, which is highly preferable for the fabrication of 3D structures. 2.2. System setup for hierarchical scaffold fabrication The high-precision hierarchical fabrication platform (Fig. 1) was composed of four sub-systems: a three-axis (XYZ) precision stage, a pneumatic dispensing unit, an EHD hot jet plotting unit, and a thermal control system. The XYZ precision stage was located on an optical table to reduce vibrational noise on which three linear stages were configured in XYZ directions with 100 nm repeatability and a displacement range of 100 × 100 × 50 mm. A high resolution camera with a maximum resolution of 0.5 m was used to monitor the deposition process. There are two deposition units for deposit PCL structures, one for pneumatic thermal extrusion, and one for EHD hot jet plotting. The temperature for both thermal extrusion and EHD hot jet plotting is kept at 82 ◦ C degree to obtain the favorable rheology of the melting polymer. The nozzle for pneumatic thermal extrusion has a 250 m ID and 450 m OD. The nozzle for EHD jet plotting has a 150 m ID and 300 m OD. The pneumatic system can provide a maximum pressure of 80 psi (550 kPa). The electrical potential between the nozzle and the substrate in EHD plotting is controlled by a high voltage source meter (TREK 610E) that can supply up to 10 K V. The temperatures of heated syringes are monitored by thermo-couple and closed-loop controlled by a temperature controller with 0.5 ◦ C degree resolution. The substrate is the glass slide rests on an aluminum coated silicon wafer that provides an electrically grounded support. The substrate and the underneath ground electrode was displaced by the XY-stage. The G-code program is used to generate the tool path of the printing system in this study. In case of complex pattern, computer-aided design (CAD) software package can be used to generate the G-code for a specific structure design then feed into the motion controller to provide the motion of the printing system. 2.3. EHD dispensing process characterization
2. Materials and methods 2.1. Materials for hybrid fabrication Polycaprolactone (PCL) pellets were purchased from Sigma–Aldrich (Milwaukee, WI) with average Mn of 45,000 g/mol
In EHD jet printing, a high voltage is applied to the nozzle tip, which causes mobile ions in the printed material (melted PCL in this study) to accumulate on the PCL/air interface. The Coulombic force causes the meniscus at the nozzle tip to deform into a conical shape (i.e., Taylor cone). With sufficiently large electric field, the
Fig. 1. Schematic of the hybrid fabrication system for E-jet printing and thermal extrusion deposition (a) EHD plotting unit. (b) Pneumatic extrusion unit. (c) The experimental testbed.
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Back Pressure Nozzle Surface Tension
Electrostatic stress
Viscous Force Whipping Region Fig. 2. Forces on Taylor cone in EHD processing.
surface charge repulsion from the electrostatic stress at the cone apex exceeds the surface tension of the meniscus, and a droplet or a jet of fluid is printed from the Taylor cone onto the grounded substrate (Fig. 2). The fluid properties of the printed material, along with the process conditions, mostly applied voltage, pressure, printing speed, and nozzle–substrate distance, determines the printing results. In this study, short nozzle–substrate distance (250 m) was chosen to utilize the stable jet region and avoid potential whipping instability. The effects of other process parameters (applied voltage, pressure, and printing speed) on the melt EHD-jet plotting process was investigated to obtain the most appropriate set of process conditions for 3D structure fabrication. 2.4. Hierarchical scaffold fabrication process The fabrication process of PCL hierarchical structures follows a layer-by-layer fashion as in Fig. 3. Step 1: A thick layer of PCL filaments with centerline space 500 m is deposited by pneumatic thermal extrusion following the programmed tool path. The process condition at this step uses the pressure of 60 psi, feedrate of 1.0 mm/s, and the nozzle-to-substrate distance of 250 m. Step 2: Micro-filament layers are dispensed by EHD hot jet plotting process onto the thick PCL structures. The orientation of
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micro-filament and thick PCL filaments can be selected by changing the plotted tool path. In this study, two orientations of the micro filaments are applied. One is along and perpendicular to the underneath thick filaments with 75 m centerline spacing. The other one is 45 and 135 degree to the underneath thick filaments with 85um spacing. This step deposits a well-connected micro filament network on the backbone thick filament structure. During EHD plotting, the pressure is chosen to be 4 psi, the feed rate is 3.0–5.0 mm/s, and the standoff height is 250 m, the applied voltage for each layer is from 1600 V to 2000 V. Step 3: Changing the orientation of the deposited structures, and repeat Step 1 and Step 2 until the desired thickness is achieved. 2.5. SEM morphology characterization The shape of the cone-jet of the electrohydrodynamic jet printing was observed by the high resolution video camera. The morphology of hierarchical scaffold structures was characterized by SEM (S-3500, Hitachi Instruments Inc., Tokyo, Japan). The scaffolds were sputter-coated with gold, and viewed at 5 kV accelerating voltage. The filament size and spacing were measured and calculated from SEM images using the image analysis software. 3. Results and discussion 3.1. Characterization of EHD hot jet plotting process In EHD printing, the applied electrical field deforms the meniscus at the nozzle tip into the Taylor cone, and eventually produces a jet from the cone shape. Generally, as the applied electric field strength increases, different electrohydrodynamic printing modes can be observed, transitioning from pulsating mode to stable jet, and then to unstable multiple jets [16]. For the melted polycaprolactone, when the nozzle to substrate gap was fixed, as we increased the voltage from 500 V, a meniscus and Taylor cone was gradually forming at the tip of the nozzle (Fig. 2a). However at a voltage lower than 800 V, there is no observed drip and jet generated from the cone. When the voltage was increased to about 825 V, a stable jet began to be ejected from the cone. As voltage increased, the stable jet remains, but both the radius and elongation of the cone became smaller (Fig. 4b and c). When the applied voltage was increased to about 1100 V unstable multi-jets appears, as shown in Fig. 4d. In our experimental condition, we did not observe any pulsating printing mode (single drop printed onto the substrate), partially due to
Fig. 3. The fabrication process of the hierarchical structures with different orientations between thick filaments and micro-filaments. (a) and (a1) Deposition of thick backbone filaments. (b) and (b1) Deposition of micro filaments network. (c) and (c1) Deposition another backbone filaments layer. (d) and (d1) Deposition of another layer of micro filaments network.
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Fig. 4. (a) Small meniscus without jet or drip at nozzle tip at 600 V. (b) and (c) Stable cone-jet at 900 V and 1050 V. (d) Unstable multi-jet at high electric field strength.
the high viscosity of the melted polycaprolactone. It is difficult for melted PCL to obtain enough electric stress to overcome the surface tension at the meniscus to form a fine drop. Obtaining stable electrohydrodynamic cone-jet mode requires the proper selection of the process parameters that include electrical voltage, pressure and plotting speed. These process conditions need to be thoroughly investigated to achieve reliable jet printing and filament formation. In this study, small nozzle–substrate gap was chosen to achieve large enough electric strength to form the stable cone-jet. At the small standoff height, the jet is kept at the stable and straight region, and the effect of whipping instability is minimized. To observe the effect of different process conditions on the cone-jet shape and the plotted filaments, one process parameter (from electrical voltage, pressure and plotting speed) was changed at a time while keeping others constant. The applied voltage played the most important role in the EHDjet printing process. Clearly there exists a lower bound threshold voltage (between 800 V and 825 V for this configuration) to continuously jet melted polymer. At a relative low voltage, a skewed cone-jet was observed as shown in Fig. 5(a1), in which the jet was inclined to the substrate along the plotting direction. As voltage increased, the skew cone-jet was changed to straight cone-jet. After the voltage was further increased, although a straight and stable
cone-jet was observed from the camera, the plotted filaments became wavy and lose controllability for precision fabrication. The amplitude of waviness increased with the increase of the voltage. This behavior can be explained by the specific material property of PCL and the jetting speed. In EHD printing, a larger voltage indicates a larger electrostatic stress, and then larger acceleration and jetting speed. When plotting speed is fixed, at the low voltage, the jetting speed was lower than the plotting speed of the stage, the high viscous force of the melted PCL deformed the cone-jet to give the skewed cone-jet. When the jetting speed matched the plotting speed, a straight cone-jet was obtained. When the jetting speed exceeds the plotting speed at high voltage, the high viscosity of the plotted and semi-solidified PCL filaments makes it difficult to reflow on the substrate. Thus the wavy filaments were obtained to absorb the over-plotted length due to speed mismatch. It is very important to select the plotting speed to match the jetting speed. The mismatch between the jetting speed and the plotting speed will significantly affect the cone-jet shape and the printed filaments. If other process parameters (voltage and pressure) are selected, the jet ejecting speed from the cone is fixed. When plotting at a small speed, wavy filaments were observed (Fig. 5(b1)). As the plotting speed increased, the filaments become straight gradually. After the plotting speed was further increased,
Fig. 5. The cone-jet shape and jet printed filaments at different conditions. (a1)–(a3) Different voltage, (b1)–(b3) different feedrate, (c1)–(c3) different pressure.
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the skewed cone-jet appeared with straight filaments left on the substrate. This specific behavior indicated the importance of matching the plotting speed with the jetting speed. For the straight filaments fabricated by the stable cone-jet and slightly skewed cone-jet, the plotting speed significantly changed the plotted filament width. The line width decreased from about 25 m to 17 m, as plotting speed increased from 1.2 mm/s to 2.4 mm/s. The results indicate that the PCL jet or semi-solidified filament can be stretched with reduced filament diameter. The plotting speed can be used as a method to adjust of the dimension of the fabricated features. It is well known that the pressure or the flow rate can affect cone-jet shape and jet stability [22]. The required pressure range for maintaining stable-jet plotting is determined by the nozzle size and other process conditions. In EHD printing, generally larger flow rate will make the jet less stable and produce whipping instability. However, for EHD-jet printing of melted PCL, we observed vibrating jet and wavy filament at the low pressure (Fig. 5(c1)). As pressure increased, the stable jet and the skewed cone-jet appeared gradually, and straight filaments were achieved. This phenomenon is again the result of the high viscosity of the melted PCL jet and interaction between the cone-jet and the printed filaments on the substrate. With a small flow rate at low pressure, a thin jet was obtained. The electrostatic stress provided large acceleration and high speed of the ejected jet. This jetting speed was larger than the plotting speed, thus wavy filaments and vibrating jet were observed. As pressure increased, large flow rate produced a thick jet. With similar electric stress (from the same voltage), less acceleration was applied to the jet, resulting smaller jetting speed. Thus the jetting speed gradually matched the plotting speed, and then was lower than the plotting speed. As a result, straight filaments and skewed jet were observed. This explanation was further supported by the increased filament width (indicating larger flow rate) along with the increase of the pressure.
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3.2. Hierarchical structure fabrication PCL is an excellent biomaterial for thermal extrusion due to its thermal stability and quick solidification. The large viscosity of melted PCL enables its direct extrusion without any supporting material. However, the large viscosity leads to the difficulty in the direct deposition of high resolution features due to the high pressure requirement. Even though higher melting temperature can reasonably lower the viscosity, the thermal stress during the solidification of melting polymer limits the usable range of the melting temperature. In the study, for the thermal extrusion of thick filaments, 180 ◦ F or 82 ◦ C is used with the pressure, feedrate and layer thickness maintained constant during the whole process at 60 psi, 1.0 mm/s and 250 m respectively. High resolution (sub-10 m) fabrication of 2D patterns and 3D structures were achieved using the EHD-jet plotting of melted polycaprolactone (Fig. 6). The smallest line width was about 5 m, which are at least one to two orders of magnitude smaller than direct melt extrusion based fabrication approaches. Because a small nozzle–substrate gap, there is less lagged jet/filament in-between the nozzle and the substrate. As a result, the EHD-jet plotting process can respond well to sharp geometric change. Fig. 6(c) and (d) demonstrates a porous 3D lattice-structured scaffold of PCL with fully interconnected inner architectures that were fabricated using the EHD hot jet plotting approach. The uniformity of the pores and the filaments indicated the applicability of using the developed EHD hot jet plotting process to fabricate structures at the micro-scale. Fabrication of hierarchical 3D scaffold structures with both large and micro filaments is very challenging, because the microfilaments need to span over a large gap (more than 250 m) without any support. Moreover, the structures deposited on the substrate essentially change the electrode geometry and charge distribution, which brings very complex perturbation to the electrostatic field
Fig. 6. High resolution two-dimensional patterns (a) and (b) and three-dimensional scaffold structure (c) and (d) fabricated by direct EHD-jet plotting of melted PCL.
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is too low, besides the winding filaments as mentioned previously, filament sagging down becomes significant, since the micro filament needs to span over more than 250 m gap. Without enough plotting speed, the jetted filaments will have severe sag down. If the plotting velocity is too high, the filaments have a large percentage for breaking up due to the thermal stress and weak mechanical structure. The effect of deposited structures on the electrostatic field strength also significantly affects the plotting performance, especially when there are an orientation angle between macrofilaments and micro-filaments. The deposited structures are acting as electrodes and will accelerate the jet around them. The jet velocity in the free space is much lower than the vicinity around the thick filaments, which is the reason why the angle on large filaments is steeper while the angle over space is smaller. It is very complicated to tune the highly coupled processing parameters. The EHD parameters for each single layer are different because the printed structures are essentially changing the collector geometry and perturb the electrostatic field and distribution. However, once these parameters are well-adjusted, the process is robust enough to obtain well-connected micro filaments network with good quality and repeatability In future, a detailed EHD plotting model will be developed to understand the EHD process and help to optimize the process parameters. 4. Conclusion
Fig. 7. SEM images of the hierarchical 3D scaffold structures.
strength and distribution, and makes the parameter tuning very difficult. During the hierarchical fabrication, we kept the standoff height between the tip and feature top surface while adjusting the applied voltage to count for the change of electrostatic field strength due to the plotted layers on the substrate. The major problem of micro filaments fabrication is their breakup when spanning over the large gap between two thick filaments. The process conditions need to be thoroughly investigated to achieve reliable jet printing and reliable filament formation. We observed that filament formation performance can be improved by decreasing feed rate and increasing the pressure, which can effectively increase micro-filament dimension for better mechanical strength. The fabricated hierarchical 3D scaffolds are shown in Fig. 7. In this study, we have fabricated two groups of scaffolds having the same macro-structure but microstructure with different orientation. The porous hierarchical 3D structures have fully interconnected inner architectures, and micro filaments are fabricated using the EHD jet plotting technology. The uniformity of pore size and the micro filaments network demonstrate the capabilities of the hybrid process for the fabrication of hierarchical scaffold structures. The hierarchical PCL scaffolds have a macro pore size of 230 ± 10 m and a porosity of 50%, with sub-10 m micro filaments, which provides the required pore size and mechanical support to allow for cell proliferation, migration, and ingrowth into the scaffolds. Particularly, the micro features can be customized to tailor the interaction between the cell and the scaffold, and provide advanced biomedical functions, such as cell alignments and cell guidance. In the hierarchical fabrication of 3D structures, choosing a proper EHD plotting speed is a critical step. When the plotting speed
In the paper, a hybrid scaffold fabrication approach is presented, which integrates electrohydrodynamic (EHD) printing technology with extrusion deposition together to fabricate 3D hierarchical scaffolds with well controlled structures at both the macro scale and micro scale. In this study, a hybrid fabrication platform and a robust fabrication process to achieve 3D hierarchical structures are developed. The melting extrusion by pneumatic pressure is used to fabricate 3D scaffolds using thermoplastic biopolymer polycaprolactone (PCL) with filaments dimension of hundreds of microns. An electrohydrodynamic (EHD) hot jet plotting process is developed to fabricate micro-scale features on the scaffolds with sub-10 m resolution, which has great potential in advanced biomedical applications, such as cell alignment and guidance. Acknowledgements The authors gratefully acknowledge support from the National Science Foundation under Grant Award CMMI 1129817 and CMMI1333775. References [1] Yang S, Leong KF, Du Z, Chua CK. The design of scaffolds for use in tissue engineering. Part I. Traditional factors. Tissue Eng 2010;7(6):679–89. [2] Sachlos E, Czernuszka JT. Making tissue engineering scaffolds work. Review: the application of solid freeform fabrication technology to the production of tissue engineering scaffolds. Eur Cell Mater 2003;5(29):39–40. [3] Tay CY, Irvine SA, Boey FY, Tan LP, Venkatraman S. Micro-/nano-engineered cellular responses for soft tissue engineering and biomedical applications. Small 2011;7:1361–78. [4] Misra SK, Ansari TI, Valappil SP, Mohn D, Philip SE, Stark WJ, et al. Poly(3hydroxybutyrate) multifunctional composite scaffolds for tissue engineering applications. Biomaterials 2010;31:2806–15. [5] Rowlands AS, Lim SA, Martin D, Cooper-White JJ. Polyurethane/poly(lacticco-glycolic) acid composite scaffolds fabricated by thermally induced phase separation. Biomaterials 2007;28:2109–21. [6] Chen W, Zhou H, Tang M, Weir MD, Bao C, Xu HHK. Gas-foaming calcium phosphate cement scaffold encapsulating human umbilical cord stem cells. Tissue Eng A 2011;18:816–27. [7] Caliari SR, Ramirez MA, Harley BA. The development of collagen-GAG scaffold-membrane composites for tendon tissue engineering. Biomaterials 2011;32(34):8990–8. [8] Hutmacher DW. Scaffolds in tissue engineering bone and cartilage. Biomaterials 2000;21(24):2529–43.
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