Article pubs.acs.org/Biomac
Cells (MC3T3-E1)-Laden Alginate Scaffolds Fabricated by a Modified Solid-Freeform Fabrication Process Supplemented with an Aerosol Spraying SeungHyun Ahn,† HyeongJin Lee,† Lawrence J. Bonassar,‡ and GeunHyung Kim*,† †
Department of Mechanical Engineering, Chosun University, GwangJu, South Korea Department of Biomedical Engineering, Cornell University, Weill Hall, Ithaca, New York, United States
‡
ABSTRACT: In this study, we propose a new cell encapsulation method consisting of a dispensing method and an aerosol-spraying method. The aerosol spray using a cross-linking agent, calcium chloride (CaCl2), was used to control the surface gelation of dispensed alginate struts during dispensing. To show the feasibility of the method, we used preosteoblast (MC3T3-E1) cells. By changing the relationship between the various dispensing/aerosol-spraying conditions and cell viability, we could determine the optimal cell-dispensing process: a nozzle size (240 μm) and an aerosol spray flow rate (0.93 ± 0.12 mL min−1), 10 mm s−1 nozzle moving speed, a 10 wt % concentration of CaCl2 in the aerosol solution, and 2 wt % concentration of CaCl2 in the second cross-linking process. Based on these optimized process conditions, we successfully fabricated a three-dimensional, porestructured, cell-laden alginate scaffold of 20 × 20 × 4.6 mm3 and 84% cell viability. During long cell culture periods (16, 25, 33, and 45 days), the preosteoblasts in the alginate scaffold survived and proliferated well.
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INTRODUCTION Cell printing, or bioprinting, is a well-known “bottom-up” technique that assembles building blocks to imitate native functional units into larger tissue constructs using a layer-bylayer approach.1 According to Guillemot et al., the technology is defined as a computer-aided process to fabricate 2D or 3D organization consisting of living cells and temporary supporting/biodegradable biomaterials for regenerating various tissues.2 Because cell printing can provide homogeneous cell spreading through whole tissue substitutes, whereas common cell-seeding techniques do not, and because one can deposit various cells in specific areas of a 3D structure mimicking tissue structure, the method can be widely applied in various tissue regenerative applications.3,4 In general, typical cell-printing methods for obtaining cell-embedded biomaterials are inkjetbased printing, laser-induced forward transfer (LIFT), and dispensing technologies.5−9 Recently, using photosensitive gelatin, two-photon polymerization combined with LIFT technology has been introduced to print multiple cell types into a pore-size controlled 3D shape.5 The cell-laden gelatin scaffold showed excellent stability in culture medium and suitability to support porcine mesenchymal stem cell adhesion and subsequent proliferation. Another inkjet bioprinting technology exhibiting spatial control of osteoblast differentiation and bone formation was described by Cooper et al.10 They printed bioink patterns of bone morphogenetic protein-2 (BMP-2) on a human allograft scaffold, and by culturing C2C12 progenitor cells, they showed that the cells were well-differentiated throughout the printed BMP-2 pattern.10 © 2012 American Chemical Society
However, although these innovative technologies have various important advantages, the techniques still have some problems to overcome. One problem is the fabrication of 3D cell-laden material thicker than 300 μm while maintaining high cell viability because for cell-laden materials of high thickness, the transportation of nutrients and waste can be limiting.11 To overcome this problem, several researchers have tried various techniques, including oxygen-producing biomaterials to reduce tissue necrosis and cellular apoptosis,12 prevascular networks in a 3D tissue construct by sandwiching endothelial cells between cell sheets to relieve hypoxia and nutrient insufficiency,13 flow perfusion culture bioreactors to improve external and internal diffusional limitations,14 and a method stacking cell-laden biopapers.11 The other approaches to overcoming the problem of cell-laden materials of high thickness include designing internal microarchitecture (pore size and porosity) in the material using a laser system with photosensitive materials and a specific mold.3,5,15 Comparisons between cell-laden materials with and without internal pore structures were made by Gaetani et al.16 Using a simple dispensing method, they printed two different types of cell-laden materials, porous and nonporous alginates with embedded human cardiac-derived cardiomyocyte progenitor cells (hCMPCs). In their study, the viability of printed cells with the porous alginate increased about 41% compared with that of the nonporous alginate at 7 days, and the porous alginate showed significant cell proliferation of hCMPCs. In terms of the internal pore structure, to achieve successfully bone tissue formation, high Received: July 19, 2012 Revised: August 20, 2012 Published: August 22, 2012 2997
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Figure 1. Schematic of the cell-encapsulation process using a dispensing method supplemented with an aerosol-spraying process with CaCl2 solution and a second cross-linking process.
under the processing conditions. Finally, the cell-embedded alginate scaffold was cultured during long periods (42 days) to observe cellular activities in the 3D alginate structure. From the perspective of cell-laden scaffolds with a precisely defined design to enable systematic studies on cell−scaffold and cell− cell interactions in a 3D structure, this method could be practically useful because various cells can be embedded readily in a 3D pore size-controllable alginate structure with this aerosol-spraying method.
porosity, appropriate pore size, and pore interconnectivity are all required, and the recommended pore size is over 300 μm, which is necessary for vascularization and the transportation of nutrients and oxygen.17 In general, to encapsulate various cells, natural and synthetic hydrogels have been widely used as artificial biomaterials. The fundamental properties of 3D printing hydrogels comprise their ability to support cellular activities including proliferation and phenotype expression, a controllable processability to allow obtaining sufficient porosity and appropriate pore size to allow the transportation of nutrients and oxygen to the cells, and the important properties of low toxicity, controllable biodegradability, and immunoprotection.18,19 Of the various hydrogels available, alginates, naturally occurring anionic polymers obtained from brown seaweeds, have been used widely for many biomedical applications due to their structural similarities to the extracellular matrices of living tissues, excellent biocompatibility, low toxicity, and easy gelation via the addition of divalent cations such as Ca2+.20,21 Additionally, because of their easy gelation, alginates can be used in the delivery of bioactive agents such as small-molecule drugs and proteins as well as in cell encapsulation.21 However, to date, no cell-laden alginate with homogeneous internal pores and completely interconnected pores has yet been fabricated due to the uncontrollable gelation process of alginates. Furthermore, high-thickness 3D cell-laden alginate structures with internal pores have not been obtained because alginates are too hydrophilic and have too low viscosity to sustain a 3D structure during the cell-dispensing process. In this study, we demonstrate an innovative cell-laden alginate with a high thickness (over 4 mm) that has controllable pore size and 100% pore interconnectivity, which was fabricated with a modified dispensing system and supplemented with an aerosol-spraying technique to provide gelation of the alginate struts during the fabrication process. To observe the effect of an aerosol-spraying method on the gelation of the alginate, we measured its rheological properties through oscillatory measurements for various aerosol-spraying conditions. Also, to detect the effects of the various dispensing conditions (nozzle size, nozzle movement speed, weight fraction of the curing agent) in the aerosol process, we measured the viability of preosteoblast cells (MC3T3-E1)
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EXPERIMENTAL SECTION
Preparation of Cell/Alginate Solution. MC3T3-E1 cells were used in the cell-printing process. They were provided by Prof. Claudia Fischbach-Teschl (Cornell University, Ithaca, NY). The cells were cultured and maintained in α-minimum essential medium (α-MEM, Life Sciences, Milwaukee, WI) containing 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO). Low-viscosity, high-G-content nonmedical-grade LF10/60 alginate (FMC BioPolymer, Drammen, Norway) was used as the cell-embedding material. We fixed the weight fraction of the alginate solution at 3.5 wt % in a phosphate-buffered saline (PBS) solution. Before loading the cells, the alginate solution was mixed with 0.5 wt % calcium chloride (CaCl2; Sigma-Aldrich) to increase the viscosity of the solution slightly, and the mixing ratio of the alginate and the CaCl2 solution was 7:3. Cells were mixed into the alginate solution using a three-way stopcock tool at a density of (1.7 to 2.3) × 106 cells mL−1. Rheological Measurements of Cell-Laden Alginate Solutions. A mixture of cells (1.7 × 106 cells mL−1) and alginate solution (3.5 wt %) was used to assess rheological properties using a rheometer (AR-2000, TA Instruments, New Castle, DE) equipped with a coneand-plate geometry (20 mm diameter; 108 μm gap; cone angle 2°). For dynamic shear measurements, dynamic frequency sweeps were conducted by applying 5% strain within the linear viscoelastic region over a frequency range between 1 and 100 rad s−1. All dynamic experiments were conducted at 27 °C. Fabrication of Cell-Laden Alginate Scaffolds. A computercontrolled three-axis robot system (DTR2-2210T, Dongbu Robot, Bucheon, South Korea) supplemented with a dispenser and an aerosol humidifier (Tess-7400; Paju, South Korea) was used for fabricating a single line of alginate and multilayered alginate structures. In the dispensing system, pneumatic pressure and the stand-off distance between a nozzle (240 μm) and working stage were fixed as 270 kPa and 220 μm, respectively. In the aerosol-spraying process, we fixed the aerosol flow rate of CaCl2 solution at 0.93 ± 0.12 mL min−1 constantly flowing onto the fabricating stage. To obtain high thickness and a 2998
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Figure 2. Rheological results for cell-embedded alginate solution (3.5 wt % alginate and 1.7 × 106 cells mL−1) with various concentrations and exposure times of CaCl2 in the aerosol-spraying solution. (a−c) Complex viscosity for various concentrations (2.5, 5, and 10 wt %) of CaCl2. (d) Comparison of complex viscosity at 1 rad s−1 for aerosol-spraying conditions. (e,f) Dynamic modulus of cell-embedded alginate sprayed with 10 wt % CaCl2 at 27 °C. (e,f) Storage (G′) modulus and loss (G″) modulus. (g) Comparison of G′ and G″ at 1 rad s−1 for various exposure times. porous, cell-laden alginate scaffold, each layer adhered to the previous one perpendicularly to form a 0°/90° strut structure. After plotting the cell-embedded multilayered alginate structure fabricated with the aerosol-spraying process and CaCl2 solution, the alginate scaffolds were cross-linked again (second curing process) with CaCl2 solution for 1 min. The detailed procedure for fabricating the cell-laden alginate scaffold is described in Figure 1. Cell Viability Measurements for Various Aerosol-Spraying Processes. After dispensing cell-laden alginate structures under various processing conditions, they were exposed to 0.15 mM calcein AM and 2 mM ethidium homodimer-1 for 45 min in an incubator. The stained cell-laden alginate structures were then analyzed under a microscope (TE2000-S; Nikon, Tokyo, Japan) equipped with an epifluorescence attachment and a SPOT RT digital camera (SPOT Imaging Solutions, Sterling Heights, MI). To observe cell viability, we captured the images and counted the numbers of green and red spots using the ImageJ software (NIH, Bethesda, MD). The viability of the cell-laden alginate scaffolds was then determined. The ratio of the number of live cells to the number of total cells, including live and
dead cells, was calculated using the software, and the ratio was normalized to the initial cell viability, the value before the cell-alginate extrusion. Initial viability was determined using trypan blue (Mediatech, Herndon, VA). In Vitro Cell Culture. The cell-laden scaffold was cultured and maintained in α-containing 10% FBS and 1% antibiotic (antimycotic; Cellgro, Mediatech, Manassas, VA). The scaffolds were incubated in an atmosphere of 5% CO2 at 37 °C, and the medium was changed every second day. After various time periods of cell culture, the scaffolds were analyzed with diamidino-2-phenylindole (DAPI) fluorescent stain to characterize the nuclei of the cells in the scaffold. Phalloidin (Invitrogen, Carlsbad, CA) was used to visualize the actin cytoskeletons of proliferated cells in the scaffolds.
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RESULTS AND DISCUSSION Rheological Properties of Alginate Solution during the Aerosol Cross-Linking Process. To obtain a porous alginate structure, we used an ionic cross-linking process with 2999
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CaCl2 because it is a common method to cross-link alginates. However, because of the its high solubility in aqueous solutions, the fast alginate gelation using CaCl2 solution has been a major drawback in its usage as a cross-linking agent; thus, when dispensing the alginate strut directly in the CaCl2 solution, the interadhesion between the dispensed alginate struts can be weakened and achieving a stable 3D layered alginate building block is difficult. To address this problem, we adjusted the aerosol-spraying process to obtain controllable gelation of the alginate struts. To observe the effects of the aerosol cross-linking process on the rheological properties of the alginate, we measured the viscoelastic responses through dynamic oscillatory measurements. To expose homogeneously the aerosol to the alginate, the alginate solution was poured in a mold (20 × 20 × 1 mm3), and the aerosol was then applied on the surface of the alginate solution. The flow rate of the aerosol process was fixed at 0.93 ± 0.12 mL min−1. Figure 2a−c shows the complex viscosity for various aerosol concentrations (2.5, 5, and 10 wt % CaCl2) and exposure times (0, 30, 60, 120, 300 s). The viscosity of the alginate solutions followed a power-law viscosity model, and although various weight fractions of CaCl2 and exposure times were applied to the alginate solution, the shear thinning behavior of the solution was similar. Figure 2d shows the variation in the increased complex viscosity of the alginate at a frequency of 1 rad s−1 as a function of the aerosol-spraying concentration of CaCl2 and various aerosol times. For the low aerosol-spraying concentration (2.5 wt % CaCl2), the alginate viscosity did not change significantly with aerosol-exposure time, indicating that the alginate was not highly cross-linked by the calcium ions. In contrast, for the high calcium concentration (10 wt % CaCl2), the viscosity of the alginate solution increased significantly as the aerosol exposure time increased. Also, the increase in the complex viscosity was primarily due to the elastic component (storage modulus, G′) rather than the viscosity component (loss modulus, G″) (Figure 2e,f). Figure 2g shows comparisons between G′ (1 rad s−1) and G″ (1 rad s−1) of alginate with various exposure times of 10 wt % CaCl2 solution. On the basis of the rheological results, we confirmed that manipulation for the gelation degree of the alginate can be controlled by altering the weight fraction and exposure time of the aerosol-spraying solution. Dispense/Aerosol Processing Parameters. To observe the dispensed alginate struts during the aerosol-spraying process, we tested the size change of a single line of an alginate strut using optical microscopy. The parameters examined were weight fraction of CaCl2 in the aerosol solution, dispensing speed, and nozzle size. For this test, the alginate solution concentration was fixed at 3.5 wt % in PBS, and to lower the effect of processing temperature on strut stability of the alginate, the temperature was fixed at 27 ± 2 °C. The flow rate of the aerosol solution was fixed at 0.93 ± 0.12 mL min−1. Figure 3 shows the effects of various weight factions of CaCl2 in the aerosol solution on the size change of the alginate strut. As shown in the graph, CaCl2 above 2.5 wt % in the aerosol solution showed negligible size reduction of a single alginate strut. We believe the concentration range of CaCl2 can provide saturated gelation of the microsized alginate strut through the aerosol process, regardless of nozzle diameter. The optical images of Figure 3a,b show the size of the dispensed alginate struts for a nozzle size of 640 μm, with and without aerosol spraying, and in the images, the scale bar = 500 μm. As shown
Figure 3. Size reduction percentage of alginate struts at various weight fractions of CaCl2 in the aerosol-spraying solution for nozzles of three diameters (240, 310, and 640 μm) moving at 10 mm s−1. Surface and cross-sectional view of a single alginate strut for (a) no-aerosol and (b) 10 wt % CaCl2 aerosol solution.
in Figure 3a, the size of the alginate strut was roughly 990 ± 32 μm for the no-aerosol-spraying process because the extruded alginate was spread out on the processing plate due to its low viscosity, whereas for the alginate strut processed with aerosol spraying (10 wt % CaCl2), the diameter of the extruded alginate strut was about 604 ± 11 μm. On the basis of the optical images, using aerosol spraying of CaCl2, the dispensed size of alginate struts was reduced significantly, about 60% for the 640 μm nozzle, indicating that the aerosol of CaCl2 can apparently cross-link the ejected alginate strut. However, by reducing the nozzle size, the size reduction percentage also decreased because the volume of dispensed alginate was lowered, so that the spreading of the extruded alginate was apparently also narrowed. In the dispensing process, the size-stability of the strut diameter is an important design factor because strut size can influence the pore size of the finally fabricated 3D structure and its mechanical properties. Therefore, we measured the diameter of the resulting alginate struts for various nozzle moving speeds (Figure 4). For a nozzle size of 240 μm and 10 wt % CaCl2 in
Figure 4. Size reduction of an alginate strut at various moving speeds of a nozzle (240 μm) under 10 weight fractions of CaCl2 in the aerosol-spraying solution.
the aerosol solution, the alginate diameter decreased significantly, like an exponential decay curve with increasing nozzle moving speed (Figure 4a−d). However, a size difference was apparent between the nozzle size (240 μm) and the alginate strut sizes that were finally obtained, likely because of the alginate solution swelling at the nozzle tip. In general, extrudate swelling is a well-known phenomenon caused by the elastic recovery of a confined macromolecule in narrow nozzle geometry (length and diameter) and shear rate. Additionally, 3000
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Figure 5. Cell (MC3T3-E1) viability for various concentrations of CaCl2 in the aerosol-spraying solution with a nozzle size of 240 μm and moving speeds (M-S) of the nozzle. Fluorescence images indicating live (green) and dead (red) cells for (a) the no-aerosol process and (b) 10 wt % CaCl2 aerosol solution.
for a speed above 20 mm s−1, the alginate strut was unstable because of the low concentration of the alginate (3.5 wt %) and too fast a moving speed of the nozzle. From this result, we selected our nozzle moving speed as 10 mm s−1 during the aerosol process. Cell Viability under Various Processing Conditions. In general, cell viability is an important parameter for cell-laden materials because viability can directly influence cellular activities, including proliferation and differentiation. To determine cell viability in the alginate structure for this aerosol process, cell-laden alginate structures were stained with calcein AM and ethidium homodimer-1 to stain live and dead cells, respectively. In fluorescence images, live cells are green and dead cells are red. To measure the effect of the weight fraction of CaCl2 in the aerosol solution on cell viability, we compared various weight fractions of CaCl2 (0, 1, 2.5, 5, and 10 wt %) at a given nozzle size, 240 μm, a moving nozzle speed of 10 mm s−1, and a pneumatic pressure of 270 kPa. Cells ((1.8 to 2.1) × 106 mL−1) were dispensed simultaneously with the alginate solution (3.5 wt %) to fabricate a one-layered structure with an aerosolspraying flow rate of 0.93 ± 0.12 mL min−1. The volume flow rate of the mixture of alginate solution and cells was 42.4 ± 3.5 μL min−1. As shown in Figure 5, for the no-aerosol process, cell viability was ∼97%, but with increasing weight fractions of CaCl2 in the aerosol process, the viability was reduced to ∼86%. However, from 2.5 to 10 wt % CaCl2 in the aerosol solution, the cell viability was similar, likely because of a saturated diffusion limitation of CaCl2 aerosol particles through the surface of the alginate struts. The fluorescence images of Figure 5 show live and dead cells in the alginate struts. As shown in Figure 5a, the alginate struts were completely spread out, like a thin polymer film on the dispensing plate, so that we could not find the structure of the alginate struts, whereas using 10 wt % CaCl2 in the aerosol spraying on the dispensed alginate struts, the alginate struts were similar to the predesigned perpendicular alginate struts. To achieve a stable pore-size-controlled, cell-laden alginate scaffold, we conducted a second cross-linking process because the aerosol process does not fully afford a stable alginate pore structure over a long-term cell culture. The cell-laden alginate structure was fabricated with the process condition of Figure 5b and immersed in three different weight fractions (1, 2, 3 wt %) of CaCl2 solution for 1 min. After the second cross-linking of the alginate structure, it was put in PBS to observe its structural stability over 2 weeks. Figure 6a−c shows the cell-embedded
Figure 6. Stability of alginate structures, fabricated with the aerosol process (10 wt % CaCl2), and a second cross-linking process (1, 2, and 3 wt % CaCl2 solution) for two time periods (1 and 2 weeks).
alginate structure in PBS. As shown in the Figures, the alginate structure cross-linked in 1 wt % CaCl2 solution collapsed completely, whereas those immersed in 2 and 3 wt % CaCl2 solution were stable over 2 weeks. From these results, we concluded that the minimum concentration of CaCl2 in the second cross-linking process is 2 wt % to obtain a stable alginate structure. To assess the effects of the second cross-linking process on cell viability, we measured the cell viability of cell-embedded alginate structures fabricated according to the process condition presented in Figure 5b. Figure 7 shows cell viability for various second cross-linking conditions with various CaCl2 solutions and the fluorescence images in Figure 7a,b display the second cross-linked alginate structures with 2 and 7 wt % CaCl2 solution, respectively. Cell viability was greatly affected by the second cross-linking process, and based on these results, we concluded that the lowest second cross-linking concentration of CaCl2, 2 wt %, showed reasonable cell viability, similar to that after the aerosol process. Through the relationships between the various processing conditions and cell viability, we fixed our cell-dispensing process: a nozzle size of 240 μm, a nozzle moving speed of 10 mm s−1, a CaCl2 concentration in the aerosol solution of 10 wt %, an aerosol-spraying flow rate of 0.93 ± 0.12 mL min−1, and a CaCl2 concentration of 2 wt % in the second cross-linking process. However, these processing conditions may differ by 3001
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Figure 7. Cell viability versus the second cross-linking process. Fluorescence images indicating live (green) and dead (red) cells for (a) 2 wt % and (b) 7 wt % CaCl2 solution under the second cross-linking condition.
cell type, weight fraction of alginate, flow rate of the aerosol, and the type of cross-linking agent. Using these optimal processing conditions, we sought to fabricate a 3D cell-embedded alginate pore structure of over 4 mm thick. Figure 8 shows an optical micrograph of the pore
cell viability in the alginate structure for surface and crosssectional areas was ∼84 ± 3%. In Vitro Cell Culture. Figure 9a−d shows optical images after 16, 25, 33, and 42 days of cell culture of the cell-embedded
Figure 9. Optical and fluorescence images for each cell culture period (16, 25, 33, and 42 days). In the fluorescence images, the nucleus (blue) and F-actin (red) are indicated. Scale bars in the optical and fluorescent images = 500 and 50 μm, respectively.
Figure 8. Three-dimensional shape of the alginate scaffold (20 × 20 × 4.6 mm3). (a) Overview optical image. (b) Surface and (c) crosssectional views, respectively. In the fluorescence image, live and dead cells are indicated by green and red, respectively.
alginate structure, and in the images, the stained nuclei (blue) and F-actin (red) in the alginate structures were readily apparent. The cells proliferated well on the alginate strut surface, indicating that the printed cells encapsulated in the alginate struts survived and increased their metabolic function during the incubation periods. Additionally, the cells with extracellular matrix (ECM) proliferated well and grew between the pores, indicating that the 3D shape and the microarchitecture of the cell-laden alginate structure were sustained during the culture period.
structure of the cell (MC3T3-E1)-laden alginate scaffold with a size of 20 × 20 × 4.6 mm3. The size of the cell-laden alginate structure was sustained, and the internal micropore structure was successfully fabricated, even though the thickness of the cell-embedded alginate scaffold was over 4 mm. As shown in the cross-sectional image, the pores in the cell-laden alginate structure were completely interconnected with channels extending uninterrupted from the top and bottom. Additionally, from the fluorescence images of the surface and crosssectional views of the structure, we found that the cells were embedded in the whole structure of the alginate structure and that the cells in the alginate struts were distributed homogenously throughout the structure. Moreover, the average
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CONCLUSIONS In this study, we propose a new cell-encapsulation method consisting of an aerosol-spraying method and solid freeform 3002
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fabrication. By controlling various processing parameters (nozzle size, nozzle moving speed, weight fraction of CaCl2 in the aerosol solution, and appropriate second cross-linking process conditions), we successfully fabricated a cell-laden alginate scaffold of 20 × 20 × 4.6 mm3, with a reasonable initial cell viability of 86%. Additionally, after several cell-culture periods (16, 25, 33, and 45 days), the preosteoblasts survived well and proliferated, and the proliferated cells and ECM gradually packed the pores and the surface of the scaffold. From the perspective of realistic 3D shapes and highly porous structures (homogeneous pore size and 100% pore interconnectivity), this method may be useful in various tissue regeneration applications.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel: +1-213-200-5649. Notes
The authors declare no competing financial interest.
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REFERENCES
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