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Flexible Fabrication of Biomimetic Bamboo-Like Hybrid Microfibers Yue Yu, Hui Wen, Jingyun Ma, Simon Lykkemark, Hui Xu, and Jianhua Qin* Fibers at micro- and nanoscale are very common in nature; for example, there are the hollow feathers on birds’ wings, knitted silks spun by spiders or silkworms, and hierarchical veins in plant leaves. These fiber materials exhibit a variety of functionalities based on their large surface area, varied configuration, flexible mechanical properties, and feasibility to be knitted into more complex structures. Inspired by these features, much effort has been devoted to the creation of artificial fiber materials for a wide range of applications in recent years. Currently, several approaches have been presented for generating fiber materials at micro- and nanoscale, including melt spinning,[1–3] wet spinning,[4–7] electrospinning,[8,9] and directwrite drawing.[10,11] These methods however can only create cylindrical fibers of a single component. Recently, some newly developed techniques have been used to synthesize fiber materials with a flexible morphology and multiple components. For example, multiple-flow compound-jet electrospinning was developed to generate titanium dioxide tubes with two to five hierarchical channels;[12] electro-hydrodynamic co-jetting was utilized for the preparation of polymer fibers with multiple compartmentalized payloads similar to Janus fibers;[13] a laminar-flowbased microfluidic approach was developed for the synthesis of a polymer microfiber with different morphologies and functions;[14-17] moreover, by controlling the spinning volume of different samples supplied via individual microchannels, microfiber or microsheets with encoded regionalized payloads were fabricated.[18,19] While these approaches have overcome the limitations of previous methods to some extent, they continue to be limited by the use of one type of base material. Nowadays, a larger challenge lies in the fabrication of biomimetic hybrid fibers with multiple types of materials, or even with complex configurations such as microscale fiber–sphere hybrid materials. Such materials may potentially exhibit multiple useful properties with flexible functionalities for a variety of new applications. In this communication, we propose a novel strategy for the one-step fabrication of biomimetic bamboo-like microscale Y. Yu, H. Wen, J. Ma, S. Lykkemark, H. Xu, Prof. J. Qin Division of Biotechnology Dalian Institute of Chemical Physics Chinese Academy of Sciences Zhongshan Road 457, Dalian, 116023, China E-mail: [email protected] S. Lykkemark Department of Clinical Medicine Aarhus University Nørrebrogade 44, 8000, Aarhus C, Denmark S. Lykkemark Sino-Danish Center for Education and Research Niels Jensens Vej 2, 8000, Aarhus C, Denmark
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hybrid fibers. By combining the droplet microfluidic technique with the wet-spinning process, biocompatible calcium alginate (CaA) microfibers can be incorporated with spherical materials, such as hydrophobic microdroplets, polymer microspheres, or multicellular spheroids. Due to the supporting effect of internal spherical materials, a unique and delicate bamboo-like architecture can be obtained. The utility of this hybrid microfiber is demonstrated by its ability to serve as a multi-functional microcarrier for different molecules, multicellular spheroids, and encoded materials. The fabrication process is characterized by its simplicity, flexibility, and controllability, demonstrating the potential of this approach for new applications in materials science and tissue engineering. In this work, a droplet microfluidic device was designed for the fabrication of a hybrid fiber (Figure 1a; experimental details are in the Supporting Information (SI)). Syringe pumps connected to the inlets were utilized to drive the oil- and aqueousphase flow in the microchannels. A spinning orifice was built at the end of the output channel for the fiber spinning process. The main functional units of the device are shown in Figure 1b. Two “T” junction geometries were used to form fluid shear force between two immiscible liquids for the generation of droplets, and the “face-to-face” design facilitated simultaneous generation of different droplets. Here, the oil phase was used as the disperse phase to form droplets by the shearing of the aqueous phase (continuous phase). Hydrophilic monomers were dissolved in the aqueous phase to form hydrogel polymer fibers. The same aqueous solution was introduced to two different continuous-phase channel paths. One was used for shearing the oil phase to form droplets (shear flow) at the “T” junction, and the other was used for adjusting the aqueousphase flow rate (compensatory flow) for the subsequent fiber spinning by supplying the aqueous solution via the side channel of the downstream cross-junction geometry. Pneumatic singlelayer membrane (SLM) valves were integrated beside the disperse phase channels. With this design, two different kinds of droplets can be incorporated simultaneously into the fiber in a highly controllable manner. This allows for the formation of fibers containing pre-defined droplet patterns. Here, alginate was chosen as the substrate for fiber fabrication due to its biocompatibility and mild gelation conditions. Prior to the fabrication of hybrid microfibers, we evaluated the performance of this platform for the generation of pure CaA microfibers alone. Initially, sodium alginate (NaA) solution was continuously introduced into the microfluidic chip from the aqueous-phase inlets using syringe pumps. The NaA solution was extruded from the microfluidic device into the solution of calcium chloride (CaCl2) via the spinning orifice. Thus, NaA was crosslinked instantly upon contact with the Ca2+ ions to form gelatinized CaA fibers. Finally, the produced fiber was
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COMMUNICATION Figure 1. Microfluidic device for the simultaneous generation of fibers and droplets, and the obtained CaA microfibers with incorporated oil droplets. a) Schematic diagram of the microfluidic chip and the generation of droplets incorporated within a fiber. b) Magnified view of the main functional units of the microfluidic chip. c) Platform used for wet-spinning and collecting fibers. d,f) Bright-field images of CaA fibers with different droplets alignments under hydrated (d) and dehydrated (f) states. e,g) Diameter and distance of the droplets in hydrated (e) and dehydrated (g) CaA fiber as functions of the disperse-phase flow rate.
collected continuously (Figure 1c). At optimal conditions, the spinning procedure exhibited good stability with flow rates between 1.0 and 10 μL/min (see Movie 1 in the SI). It is noted that, due to the wet-spinning principle, the diameter of the obtained fiber mainly depends on the size of the spinning orifice rather than the flow rate. In contrast to the sheath flow method, the presented fiber generation process is very simple and convenient, which greatly facilitates the subsequent incorporation of spherical materials with enhanced functionality. After the stable formation of the hydrogel CaA fiber, we further explored the possibility of fabricating hybrid fibers by incorporating different spherical materials. It is known that the droplet microfluidic approach provides an ideal method for the fabrication of monodisperse microspheres, which are commonly used for various applications in materials synthesis. Herein, we made attempts to combine the droplet microfluidic approach with the wet-spinning process in order to produce hybrid materials with different properties and configurations. Because the formation of droplets is the basis of the synthesis of spherical materials, hydrophobic droplets were initially generated and incorporated into a CaA microfiber using the droplet microfluidic device as described above. A hydrophobic liquid (fluorinated oil, FC-40) was used as the oil phase to form monodisperse droplets in NaA solution by utilizing one of the “T” junctions. The NaA solution was immediately crosslinked to form gelatinized CaA fiber after contact with the CaCl2 solution in the bath. Thus, the continuous generation of ordered
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droplets and gelation of microfibers happened simultaneously (see Movie 2 and Figure S1 in the SI). Based on this process, hybrid microfibers with diverse morphology could be produced under hydrated and dehydrated conditions (Figure 1d,f). In particular, under dehydrated conditions, a necklace-like morphology resulted, in which a set of “pearls” (aligned droplets) were “on a thread” (CaA fiber), with a maximum droplet-diameter-to-fiber-thickness ratio of 10:3 (Figure 1f, top). The formation of this morphology is mainly dependent on the supporting effects of the inner droplets and a sufficient distance between the aligned droplets, which is controlled by the disperse-phase flow rate. It is clear that the morphology of the fibers is greatly affected by both diameter and distance between the ordered droplets. In addition, the flow rate of FC-40 is a critical factor in determining the alignments of the droplets within the microfiber under a fixed continuous-phase flow rate, and its effects on the droplets size and distance within the microfiber are shown in Figure 1e and g. The results imply that an increase of the disperse-phase flow significantly decreases the internal distance between the droplets within the CaA microfiber with only minor effects on droplet size. It is predicted that the hybrid microfibers containing aligned droplets may be a unique way to produce high-throughput microreactors in fibers for various biological and chemical reactions, without having to consider sample contamination caused by droplet coalescence. Based on the incorporated hydrophobic droplets, we further explored the feasibility of fabricating CaA microfibers hybridized
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Figure 2. Incorporation of PLGA spheres and cell spheroids in CaA microfiber. a) Schematic diagram illustrating the principle of synthesizing PLGA microspheres hybridized within a CaA microfiber. PDMS represents polydimethylsiloxane. b) Images of hybrid CaA fibers in the hydrated state. c) Images of the biomimetic bamboo-like hybrid microfibers in dehydrated state. The green spheroids are PLGA particles stained by fluorescein (GFP). d) Brightfield image of CaA fibers with encapsulated mesenchymal stem cell (MSC) spheroids. e,f) Fluorescent images of closely aligned MSC spheroids in CaA fibers. g) Confocal image of encapsulated MSC spheroids in fiber.
with polymer microspheres. Here, polymer poly(lactic-co-glycolic acid) (PLGA) was selected to be manipulated into spherical configuration and incorporated into a CaA microfiber. PLGA has been used extensively as a biocompatible and biodegradable material in drug delivery and tissue engineering. The microsphere formation and incorporation process was realized based on an emulsification and solvent rapid evaporation principle (Figure 2a). Initially, PLGA was dissolved in dimethyl carbonate (DMC) to serve as the oil phase. The PLGA-DMC mixture was emulsified to form droplets in NaA solution (droplet generation). Afterwards, the NaA was gelatinized to form a CaA microfiber with internal PLGA-DMC droplets (alginate gelatinization) by the method described above. Meanwhile, the DMC solvent evaporated rapidly out of the fiber material (solvent evaporation), causing the PLGA-DMC droplets to shrink dramatically and form solidified PLGA microspheres within the microfiber (Figure 2b). Considering the droplets’ template
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effect, we expected to see spherical cavities surrounding the PLGA microspheres within the CaA microfiber under the hydrated state. Instead, the produced cavities exhibited a bulletlike shape (Figure 2b). We assume that this might be caused by the following reasons. In the wet-spinning process, NaA solution passes through the spinning orifice in which the flow rate of central stream is faster than that of the marginal stream; at the same time, the gelation process occurs from the outside towards the inside of the stream. Thus, after the fiber is spun out of the device, the internal droplets can still move forward some distance in the uncured NaA solution accompanying with shrinking process, leading to the generation of “bullet-like” cavities. It is worth noting that the organic solvent DMC plays a critical role in fabricating the polymer microspheres in microfibers. Its hydrophobic properties facilitate the formation of PLGADMC droplets, and the highly volatile nature of the solvent enables solidification of PLGA within a very short time under
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bioreactor for the in-vitro simulation of the in-vivo growth of microtissues. The fiber-supported cell aggregates can protect cells from shear stress and allow a sufficient supply of oxygen and nutrients, resulting in good viability. Moreover, this type of carrier culture is accommodated with higher cell densities than those obtained in the 2D dish culture, therefore simplifying the scale-up process. Consequently, for some rare cells such as stem cells, this type of cultivation has a tremendous advantage owing to the scaled-up cell investigation accompanied by the lower cost due to a reduction in the amount of growth factors, media, and other expensive supplements. In addition, we envision that the multiple fibers bearing cell spheroids can be further intertwined to investigate the interactions between aligned spheroids or cell–cell communication among the spheroids located in different positions of the fibers. Furthermore, the cell spheroids can reconstitute intrinsic morphologies and functions of some living tissues, and the bio-hybrid microfiber can be used as a highly effective carrier for transplantation of biomimetic micro-tissues in regenerative medicine. As a biological storage, cell spheroids can be released from dissolved CaA microfiber simply by using the proper media, which might facilitate the stem cell research and tissue engineering applications. As described above, we have successfully fabricated CaA microfibers with various spherical materials such as hydrophobic droplets, PLGA spheres, and MSC spheroids. In addition, more components can be encoded into CaA microfiber simultaneously by utilizing the SLM valves in a controllable way. We predict that the increased complexity may endow the fiber materials even more flexibility and functionality. Herein, we have made attempts to encode two kinds of hydrophobic droplets into the CaA microfiber. As shown in Figure 3a, the digital controller was composed of a gas cylinder and solenoid valve equipment. Customized software was developed to control the solenoid valves, thereby regulating the SLM valves. By digitally activating the valves as shown in Figure 3b, the oil-phase channel was deformed, and the oil liquid was squeezed out to form droplets in a highly controllable manner (see Movie 3, SI). We found that the activated frequency of SLM valves and the flow rate of the dispersed phase have substantial effects on the size of generated droplets within the microfiber. The results in Figure 3c implied that the size of FC-40 droplets decreased with the increased frequency of the active valves. As a case study, different n-hexadecane droplets stained by fluorescein and rhodamine B were encoded into the CaA microfiber. Under the control of a digital controller, the SLM valves were activated alternately in a highly ordered manner via prescribed programs as shown in Figure 3d. CaA microfibers with four droplets encoded patterns, including “-A-B-”, “-A-A-B-”, “-A-A-B-B-”, and “-A-A-BA-B” (“A” represents n-hexadecane stained by rhodamine B, “B” represents n-hexadecane stained by fluorescein) were obtained, which are in accordance with the program settings (Figure 3e). The achieved encoding process demonstrated the feasibility to fabricate versatile hybrid fiber materials using this platform. In summary, we proposed a novel droplet microfluidic approach combined with a wet-spinning process for the one-step fabrication of a biomimetic hybrid fiber with multiple functionalities at microscale. Various spherical materials with different properties like hydrophobic microdroplets, polymer microspheres, and cell microspheroids were incorporated into the CaA microfiber in
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aqueous solution. Moreover, the obtained CaA microfiber with PLGA spheres can be shrunk to biomimetic bamboo-like architecture by dehydrating (Figure 2c). This hybrid material is composed of a CaA microfiber stem tightened around the aligned polymer microspheres, which resembles the joints on bamboo. In contrast to previously reported works, this hybrid material combines different types of materials with diverse properties and configurations in one entity. To our knowledge, the fabrication of such hybrid fiber materials has never been reported. Owing to the combination of materials embracing a diverse set of characteristics, the hybrid fiber is expected to exhibit not only good biocompatibility and biodegradability, but also an enhanced multifunctionality. Actually, the microspheres– microfiber hybrid is expected to serve as a dual delivery carrier. Both Hydrophilic and hydrophobic molecules can be loaded to hydrogel CaA microfibers and polymer PLGA microspheres, respectively, leading to independent release of those molecules from the hybrid fiber. Thus, the release kinetics of each type of molecule can be regulated individually and easily by adjusting the properties of the fiber materials such as the molecular weight or the concentration of the encapsulated molecules. In addition, we previously made use of the double emulsion PLGA-DMC droplets with an inner aqueous phase to fabricate a honeycomb material.[20] Here, if the present single-emulsion droplets (O/W, oil/water) are replaced with the double-emulsion PLGA-DMC droplets (W/O/W), even more types of molecules can be loaded into the inner aqueous phase, middle PLGA phase, or outer CaA phase. This hybrid microfiber may exhibit potential utility as a microcarrier for synergistically functional molecules in biological studies. In the following work, we further explored the biofunctionality of the hybrid materials by encapsulating living multicellular aggregates derived from mesenchymal stem cells (MSCs). MSCs are progenitor cells possessing high capacity of selfrenewal while maintaining multipotency to differentiate into a variety of cell types, and are therefore considered to have enormous therapeutic potential for tissue repair. Prior to the encapsulation process, MSC spheroids were initially formed based on a concave-well array chip.[21] The generated MSC spheroids were collected and suspended in NaA solution, which was further applied for the generation of hybrid CaA microfibers by using the same platform as described above. The newly formed biohybrid microfibers with encapsulated multicellular spheroids are shown in Figure 2d. The microfibers transferred to cell culture medium could maintain long-term cultivation with sufficient nutrients and oxygen exchange. During the cultivation, the encapsulated MSC spheroids in 3D microenvironment exhibited good viability and proliferation ability. Figure 2e and f shows the fluorescence images of the microfibers encapsulated with aligned green fluorescent protein (GFP)-transfected MSC spheroids with 4′,6-diamidino-2-phenylindole (DAPI) nuclear counterstain, and Figure 2g shows an enlarged confocal image of a single MSC spheroid within a microfiber. It is generally accepted that multicellular spheroids are an ideal model for the simulation of a 3D physiologically relevant microenvironment in vitro, which can be used in the evaluation of the physiological response to therapeutic compounds and to facilitate more accurate drug evaluations. Here, the integration of CaA microfibers and MSC spheroids affords a powerful
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Figure 3. Programmable droplets encoding in CaA fiber. a) Schematic diagram shows the constitution of droplets encoding platform. b) Bright field image of the top view and schematic diagram of the cross-section view of the SLM valve. Two different states (inactivated state and activated state) are shown. c) Valve-activated frequency and dispersed-phase flow rate as function of droplets radius. d) Schematic description of the droplets encoding program setting. e) Confocal images show a dehydrated CaA fiber encoded by two different droplets (red and green). Inserted are bright-field images of the fibers with droplets (lower left) and minimal repeating units of the encoded droplets.
a controllable manner. The novelty of this new approach lies in its simplicity, flexibility, and controllability in the fabrication of hybrid fiber–sphere materials, which is not possible by other approaches. Benefiting from the variety in composition, configuration, and biocompatibility, these types of biomimetic hybrid fiber materials hold great promise for applications in materials science, tissue engineering, and regenerative medicine.
Foundation of China (No. 91227123), Key Projects in the National Science & Technology Pillar Program in the Twelfth Five-Year Plan Period of China (No. 2012BAK02B00 and 2012BAK02B03), the Special Fund for Agro-scientific Research in the Public Interest (No. 201303045). Received: October 6, 2013 Revised: November 13, 2013 Published online: January 22, 2014
Experimental Section See supporting information.
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements This research was supported by the Joint Research Fund of NSFC-RGC (11161160552, N_HKUST601/11), the National Nature Science
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[1] K. Mezghani, J. E. Spuriell, J. Polym. Sci. B: Polym. Phys. 1998, 36, 1005. [2] L. Fambri, A. Pegorretti, R. Fenner, S. D. Incardona, C. Migliaresi, Polymer 1997, 38, 79. [3] P. Pötschke, H. Brünig, A. Janke, D. Fischer, D. Jehnichen, Polymer 2005, 46, 10355. [4] S. Hirano, Macromol. Symp. 2001, 168, 21. [5] K. D. Nelson, A. Romero, P. Waggoner, B. Crow, A. Borneman, G. M. Smith, Tissue Eng. 2003, 9, 1323. [6] W. Qiu, W. Teng, J. Cappello, X. Wu, Biomacromolecules 2009, 10, 602. [7] S. Zhang, K. K. K. Koziol, I. A. Kinloch, A. H. Windle, Small 2008, 4, 1217. [8] A. Greiner, J. H. Wendorff, J. Am. Chem. Soc. 2007, 46, 5670.
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[16] J. H. Jung, C. H. Choi, S. Chung, Y. M. Chung, C. S. Lee, Lab Chip 2009, 9, 2596. [17] J. K. Nunes, H. Constantin, H. A. Stone, Soft Matter 2013, 9, 4227. [18] E. Kang, G. S. Jeong, Y. Y. Choi, K. H. Lee, A. Khademhosseini, S. H. Lee, Nat. Mater. 2011, 10, 877. [19] L. Leng, A. McAllister, B. Zhang, M. Radisic, A. Günther, Adv. Mater. 2012, 24, 3650. [20] J. Ma, Y. S. Hui, M. Zhang, Y. Yu, W. Wen, J. Qin, Small 2013, 9, 497. [21] J. Y. Park, D. H. Lee, E. J. Lee, S. H. Lee, Lab Chip 2009, 9, 2043.
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[9] T. J. Sill, H. A. von Recum, Biomaterials 2008, 29, 1989. [10] S. M. Berry, S. Pabba, J. Crest, S. D. Cambron, G. H. McKinley, R. W. Cohn, R. S. Keynton, Polymer 2011, 52, 1654. [11] S. M. Berry, S. P. Warren, D. A. Hilgart, A. T. Schworer, S. Pabba, A. S. Gobin, R. W. Cohn, R. S. Keynton, Biomaterials 2011, 32, 1872. [12] Y. Zhao, X. Cao, L. Jiang, J. Am. Chem. Soc. 2007, 129, 764. [13] J. Lahann, Small 2011, 7, 1149. [14] D. A. Boyd, A. R. Shields, P. B. Howell, F. S. Ligler, Lab Chip 2013, 13, 3105. [15] C. H. Choi, H. Yi, S. Hwang, D. A. Weitz, C. S. Lee, Lab Chip 2011, 11, 1477.
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Flexible Fabrication of Biomimetic Bamboo-Like Hybrid Microfibers Yue Yu, Hui Wen, Jingyun Ma, Simon Lykkemark, Hui Xu, and Jianhua Qin* Fibers at micro- and nanoscale are very common in nature; for example, there are the hollow feathers on birds’ wings, knitted silks spun by spiders or silkworms, and hierarchical veins in plant leaves. These fiber materials exhibit a variety of functionalities based on their large surface area, varied configuration, flexible mechanical properties, and feasibility to be knitted into more complex structures. Inspired by these features, much effort has been devoted to the creation of artificial fiber materials for a wide range of applications in recent years. Currently, several approaches have been presented for generating fiber materials at micro- and nanoscale, including melt spinning,[1–3] wet spinning,[4–7] electrospinning,[8,9] and directwrite drawing.[10,11] These methods however can only create cylindrical fibers of a single component. Recently, some newly developed techniques have been used to synthesize fiber materials with a flexible morphology and multiple components. For example, multiple-flow compound-jet electrospinning was developed to generate titanium dioxide tubes with two to five hierarchical channels;[12] electro-hydrodynamic co-jetting was utilized for the preparation of polymer fibers with multiple compartmentalized payloads similar to Janus fibers;[13] a laminar-flowbased microfluidic approach was developed for the synthesis of a polymer microfiber with different morphologies and functions;[14-17] moreover, by controlling the spinning volume of different samples supplied via individual microchannels, microfiber or microsheets with encoded regionalized payloads were fabricated.[18,19] While these approaches have overcome the limitations of previous methods to some extent, they continue to be limited by the use of one type of base material. Nowadays, a larger challenge lies in the fabrication of biomimetic hybrid fibers with multiple types of materials, or even with complex configurations such as microscale fiber–sphere hybrid materials. Such materials may potentially exhibit multiple useful properties with flexible functionalities for a variety of new applications. In this communication, we propose a novel strategy for the one-step fabrication of biomimetic bamboo-like microscale Y. Yu, H. Wen, J. Ma, S. Lykkemark, H. Xu, Prof. J. Qin Division of Biotechnology Dalian Institute of Chemical Physics Chinese Academy of Sciences Zhongshan Road 457, Dalian, 116023, China E-mail: [email protected] S. Lykkemark Department of Clinical Medicine Aarhus University Nørrebrogade 44, 8000, Aarhus C, Denmark S. Lykkemark Sino-Danish Center for Education and Research Niels Jensens Vej 2, 8000, Aarhus C, Denmark
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hybrid fibers. By combining the droplet microfluidic technique with the wet-spinning process, biocompatible calcium alginate (CaA) microfibers can be incorporated with spherical materials, such as hydrophobic microdroplets, polymer microspheres, or multicellular spheroids. Due to the supporting effect of internal spherical materials, a unique and delicate bamboo-like architecture can be obtained. The utility of this hybrid microfiber is demonstrated by its ability to serve as a multi-functional microcarrier for different molecules, multicellular spheroids, and encoded materials. The fabrication process is characterized by its simplicity, flexibility, and controllability, demonstrating the potential of this approach for new applications in materials science and tissue engineering. In this work, a droplet microfluidic device was designed for the fabrication of a hybrid fiber (Figure 1a; experimental details are in the Supporting Information (SI)). Syringe pumps connected to the inlets were utilized to drive the oil- and aqueousphase flow in the microchannels. A spinning orifice was built at the end of the output channel for the fiber spinning process. The main functional units of the device are shown in Figure 1b. Two “T” junction geometries were used to form fluid shear force between two immiscible liquids for the generation of droplets, and the “face-to-face” design facilitated simultaneous generation of different droplets. Here, the oil phase was used as the disperse phase to form droplets by the shearing of the aqueous phase (continuous phase). Hydrophilic monomers were dissolved in the aqueous phase to form hydrogel polymer fibers. The same aqueous solution was introduced to two different continuous-phase channel paths. One was used for shearing the oil phase to form droplets (shear flow) at the “T” junction, and the other was used for adjusting the aqueousphase flow rate (compensatory flow) for the subsequent fiber spinning by supplying the aqueous solution via the side channel of the downstream cross-junction geometry. Pneumatic singlelayer membrane (SLM) valves were integrated beside the disperse phase channels. With this design, two different kinds of droplets can be incorporated simultaneously into the fiber in a highly controllable manner. This allows for the formation of fibers containing pre-defined droplet patterns. Here, alginate was chosen as the substrate for fiber fabrication due to its biocompatibility and mild gelation conditions. Prior to the fabrication of hybrid microfibers, we evaluated the performance of this platform for the generation of pure CaA microfibers alone. Initially, sodium alginate (NaA) solution was continuously introduced into the microfluidic chip from the aqueous-phase inlets using syringe pumps. The NaA solution was extruded from the microfluidic device into the solution of calcium chloride (CaCl2) via the spinning orifice. Thus, NaA was crosslinked instantly upon contact with the Ca2+ ions to form gelatinized CaA fibers. Finally, the produced fiber was
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COMMUNICATION Figure 1. Microfluidic device for the simultaneous generation of fibers and droplets, and the obtained CaA microfibers with incorporated oil droplets. a) Schematic diagram of the microfluidic chip and the generation of droplets incorporated within a fiber. b) Magnified view of the main functional units of the microfluidic chip. c) Platform used for wet-spinning and collecting fibers. d,f) Bright-field images of CaA fibers with different droplets alignments under hydrated (d) and dehydrated (f) states. e,g) Diameter and distance of the droplets in hydrated (e) and dehydrated (g) CaA fiber as functions of the disperse-phase flow rate.
collected continuously (Figure 1c). At optimal conditions, the spinning procedure exhibited good stability with flow rates between 1.0 and 10 μL/min (see Movie 1 in the SI). It is noted that, due to the wet-spinning principle, the diameter of the obtained fiber mainly depends on the size of the spinning orifice rather than the flow rate. In contrast to the sheath flow method, the presented fiber generation process is very simple and convenient, which greatly facilitates the subsequent incorporation of spherical materials with enhanced functionality. After the stable formation of the hydrogel CaA fiber, we further explored the possibility of fabricating hybrid fibers by incorporating different spherical materials. It is known that the droplet microfluidic approach provides an ideal method for the fabrication of monodisperse microspheres, which are commonly used for various applications in materials synthesis. Herein, we made attempts to combine the droplet microfluidic approach with the wet-spinning process in order to produce hybrid materials with different properties and configurations. Because the formation of droplets is the basis of the synthesis of spherical materials, hydrophobic droplets were initially generated and incorporated into a CaA microfiber using the droplet microfluidic device as described above. A hydrophobic liquid (fluorinated oil, FC-40) was used as the oil phase to form monodisperse droplets in NaA solution by utilizing one of the “T” junctions. The NaA solution was immediately crosslinked to form gelatinized CaA fiber after contact with the CaCl2 solution in the bath. Thus, the continuous generation of ordered
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droplets and gelation of microfibers happened simultaneously (see Movie 2 and Figure S1 in the SI). Based on this process, hybrid microfibers with diverse morphology could be produced under hydrated and dehydrated conditions (Figure 1d,f). In particular, under dehydrated conditions, a necklace-like morphology resulted, in which a set of “pearls” (aligned droplets) were “on a thread” (CaA fiber), with a maximum droplet-diameter-to-fiber-thickness ratio of 10:3 (Figure 1f, top). The formation of this morphology is mainly dependent on the supporting effects of the inner droplets and a sufficient distance between the aligned droplets, which is controlled by the disperse-phase flow rate. It is clear that the morphology of the fibers is greatly affected by both diameter and distance between the ordered droplets. In addition, the flow rate of FC-40 is a critical factor in determining the alignments of the droplets within the microfiber under a fixed continuous-phase flow rate, and its effects on the droplets size and distance within the microfiber are shown in Figure 1e and g. The results imply that an increase of the disperse-phase flow significantly decreases the internal distance between the droplets within the CaA microfiber with only minor effects on droplet size. It is predicted that the hybrid microfibers containing aligned droplets may be a unique way to produce high-throughput microreactors in fibers for various biological and chemical reactions, without having to consider sample contamination caused by droplet coalescence. Based on the incorporated hydrophobic droplets, we further explored the feasibility of fabricating CaA microfibers hybridized
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Figure 2. Incorporation of PLGA spheres and cell spheroids in CaA microfiber. a) Schematic diagram illustrating the principle of synthesizing PLGA microspheres hybridized within a CaA microfiber. PDMS represents polydimethylsiloxane. b) Images of hybrid CaA fibers in the hydrated state. c) Images of the biomimetic bamboo-like hybrid microfibers in dehydrated state. The green spheroids are PLGA particles stained by fluorescein (GFP). d) Brightfield image of CaA fibers with encapsulated mesenchymal stem cell (MSC) spheroids. e,f) Fluorescent images of closely aligned MSC spheroids in CaA fibers. g) Confocal image of encapsulated MSC spheroids in fiber.
with polymer microspheres. Here, polymer poly(lactic-co-glycolic acid) (PLGA) was selected to be manipulated into spherical configuration and incorporated into a CaA microfiber. PLGA has been used extensively as a biocompatible and biodegradable material in drug delivery and tissue engineering. The microsphere formation and incorporation process was realized based on an emulsification and solvent rapid evaporation principle (Figure 2a). Initially, PLGA was dissolved in dimethyl carbonate (DMC) to serve as the oil phase. The PLGA-DMC mixture was emulsified to form droplets in NaA solution (droplet generation). Afterwards, the NaA was gelatinized to form a CaA microfiber with internal PLGA-DMC droplets (alginate gelatinization) by the method described above. Meanwhile, the DMC solvent evaporated rapidly out of the fiber material (solvent evaporation), causing the PLGA-DMC droplets to shrink dramatically and form solidified PLGA microspheres within the microfiber (Figure 2b). Considering the droplets’ template
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effect, we expected to see spherical cavities surrounding the PLGA microspheres within the CaA microfiber under the hydrated state. Instead, the produced cavities exhibited a bulletlike shape (Figure 2b). We assume that this might be caused by the following reasons. In the wet-spinning process, NaA solution passes through the spinning orifice in which the flow rate of central stream is faster than that of the marginal stream; at the same time, the gelation process occurs from the outside towards the inside of the stream. Thus, after the fiber is spun out of the device, the internal droplets can still move forward some distance in the uncured NaA solution accompanying with shrinking process, leading to the generation of “bullet-like” cavities. It is worth noting that the organic solvent DMC plays a critical role in fabricating the polymer microspheres in microfibers. Its hydrophobic properties facilitate the formation of PLGADMC droplets, and the highly volatile nature of the solvent enables solidification of PLGA within a very short time under
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bioreactor for the in-vitro simulation of the in-vivo growth of microtissues. The fiber-supported cell aggregates can protect cells from shear stress and allow a sufficient supply of oxygen and nutrients, resulting in good viability. Moreover, this type of carrier culture is accommodated with higher cell densities than those obtained in the 2D dish culture, therefore simplifying the scale-up process. Consequently, for some rare cells such as stem cells, this type of cultivation has a tremendous advantage owing to the scaled-up cell investigation accompanied by the lower cost due to a reduction in the amount of growth factors, media, and other expensive supplements. In addition, we envision that the multiple fibers bearing cell spheroids can be further intertwined to investigate the interactions between aligned spheroids or cell–cell communication among the spheroids located in different positions of the fibers. Furthermore, the cell spheroids can reconstitute intrinsic morphologies and functions of some living tissues, and the bio-hybrid microfiber can be used as a highly effective carrier for transplantation of biomimetic micro-tissues in regenerative medicine. As a biological storage, cell spheroids can be released from dissolved CaA microfiber simply by using the proper media, which might facilitate the stem cell research and tissue engineering applications. As described above, we have successfully fabricated CaA microfibers with various spherical materials such as hydrophobic droplets, PLGA spheres, and MSC spheroids. In addition, more components can be encoded into CaA microfiber simultaneously by utilizing the SLM valves in a controllable way. We predict that the increased complexity may endow the fiber materials even more flexibility and functionality. Herein, we have made attempts to encode two kinds of hydrophobic droplets into the CaA microfiber. As shown in Figure 3a, the digital controller was composed of a gas cylinder and solenoid valve equipment. Customized software was developed to control the solenoid valves, thereby regulating the SLM valves. By digitally activating the valves as shown in Figure 3b, the oil-phase channel was deformed, and the oil liquid was squeezed out to form droplets in a highly controllable manner (see Movie 3, SI). We found that the activated frequency of SLM valves and the flow rate of the dispersed phase have substantial effects on the size of generated droplets within the microfiber. The results in Figure 3c implied that the size of FC-40 droplets decreased with the increased frequency of the active valves. As a case study, different n-hexadecane droplets stained by fluorescein and rhodamine B were encoded into the CaA microfiber. Under the control of a digital controller, the SLM valves were activated alternately in a highly ordered manner via prescribed programs as shown in Figure 3d. CaA microfibers with four droplets encoded patterns, including “-A-B-”, “-A-A-B-”, “-A-A-B-B-”, and “-A-A-BA-B” (“A” represents n-hexadecane stained by rhodamine B, “B” represents n-hexadecane stained by fluorescein) were obtained, which are in accordance with the program settings (Figure 3e). The achieved encoding process demonstrated the feasibility to fabricate versatile hybrid fiber materials using this platform. In summary, we proposed a novel droplet microfluidic approach combined with a wet-spinning process for the one-step fabrication of a biomimetic hybrid fiber with multiple functionalities at microscale. Various spherical materials with different properties like hydrophobic microdroplets, polymer microspheres, and cell microspheroids were incorporated into the CaA microfiber in
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aqueous solution. Moreover, the obtained CaA microfiber with PLGA spheres can be shrunk to biomimetic bamboo-like architecture by dehydrating (Figure 2c). This hybrid material is composed of a CaA microfiber stem tightened around the aligned polymer microspheres, which resembles the joints on bamboo. In contrast to previously reported works, this hybrid material combines different types of materials with diverse properties and configurations in one entity. To our knowledge, the fabrication of such hybrid fiber materials has never been reported. Owing to the combination of materials embracing a diverse set of characteristics, the hybrid fiber is expected to exhibit not only good biocompatibility and biodegradability, but also an enhanced multifunctionality. Actually, the microspheres– microfiber hybrid is expected to serve as a dual delivery carrier. Both Hydrophilic and hydrophobic molecules can be loaded to hydrogel CaA microfibers and polymer PLGA microspheres, respectively, leading to independent release of those molecules from the hybrid fiber. Thus, the release kinetics of each type of molecule can be regulated individually and easily by adjusting the properties of the fiber materials such as the molecular weight or the concentration of the encapsulated molecules. In addition, we previously made use of the double emulsion PLGA-DMC droplets with an inner aqueous phase to fabricate a honeycomb material.[20] Here, if the present single-emulsion droplets (O/W, oil/water) are replaced with the double-emulsion PLGA-DMC droplets (W/O/W), even more types of molecules can be loaded into the inner aqueous phase, middle PLGA phase, or outer CaA phase. This hybrid microfiber may exhibit potential utility as a microcarrier for synergistically functional molecules in biological studies. In the following work, we further explored the biofunctionality of the hybrid materials by encapsulating living multicellular aggregates derived from mesenchymal stem cells (MSCs). MSCs are progenitor cells possessing high capacity of selfrenewal while maintaining multipotency to differentiate into a variety of cell types, and are therefore considered to have enormous therapeutic potential for tissue repair. Prior to the encapsulation process, MSC spheroids were initially formed based on a concave-well array chip.[21] The generated MSC spheroids were collected and suspended in NaA solution, which was further applied for the generation of hybrid CaA microfibers by using the same platform as described above. The newly formed biohybrid microfibers with encapsulated multicellular spheroids are shown in Figure 2d. The microfibers transferred to cell culture medium could maintain long-term cultivation with sufficient nutrients and oxygen exchange. During the cultivation, the encapsulated MSC spheroids in 3D microenvironment exhibited good viability and proliferation ability. Figure 2e and f shows the fluorescence images of the microfibers encapsulated with aligned green fluorescent protein (GFP)-transfected MSC spheroids with 4′,6-diamidino-2-phenylindole (DAPI) nuclear counterstain, and Figure 2g shows an enlarged confocal image of a single MSC spheroid within a microfiber. It is generally accepted that multicellular spheroids are an ideal model for the simulation of a 3D physiologically relevant microenvironment in vitro, which can be used in the evaluation of the physiological response to therapeutic compounds and to facilitate more accurate drug evaluations. Here, the integration of CaA microfibers and MSC spheroids affords a powerful
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Figure 3. Programmable droplets encoding in CaA fiber. a) Schematic diagram shows the constitution of droplets encoding platform. b) Bright field image of the top view and schematic diagram of the cross-section view of the SLM valve. Two different states (inactivated state and activated state) are shown. c) Valve-activated frequency and dispersed-phase flow rate as function of droplets radius. d) Schematic description of the droplets encoding program setting. e) Confocal images show a dehydrated CaA fiber encoded by two different droplets (red and green). Inserted are bright-field images of the fibers with droplets (lower left) and minimal repeating units of the encoded droplets.
a controllable manner. The novelty of this new approach lies in its simplicity, flexibility, and controllability in the fabrication of hybrid fiber–sphere materials, which is not possible by other approaches. Benefiting from the variety in composition, configuration, and biocompatibility, these types of biomimetic hybrid fiber materials hold great promise for applications in materials science, tissue engineering, and regenerative medicine.
Foundation of China (No. 91227123), Key Projects in the National Science & Technology Pillar Program in the Twelfth Five-Year Plan Period of China (No. 2012BAK02B00 and 2012BAK02B03), the Special Fund for Agro-scientific Research in the Public Interest (No. 201303045). Received: October 6, 2013 Revised: November 13, 2013 Published online: January 22, 2014
Experimental Section See supporting information.
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements This research was supported by the Joint Research Fund of NSFC-RGC (11161160552, N_HKUST601/11), the National Nature Science
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