Creating Hierarchical Topographies on Fibrous Platforms Using ...

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Creating Hierarchical Topographies on Fibrous Platforms Using Femtosecond Laser Ablation for Directing Myoblasts Behavior Indong Jun,† Yong-Woo Chung,†,§ Yun-Hoe Heo,∥,⊥ Hyung-Seop Han,† Jimin Park,† Hongsoo Jeong,†,§ Hyunjung Lee,† Yu Bin Lee,∥,⊥ Yu-Chan Kim,†,‡ Hyun-Kwang Seok,†,‡ Heungsoo Shin,*,∥,⊥ and Hojeong Jeon*,†,‡ †

Center for Biomaterials, Biomedical Research Institute, Korea Institute of Science & Technology, Seoul 02792, Republic of Korea Korea University of Science and Technology, Daejeon 34113, Republic of Korea § Department of Mechanical Engineering, Korea University, Seoul 02841, Republic of Korea ∥ Department of Bioengineering, Hanyang University, Seoul 04763, Republic of Korea ⊥ BK21 Plus Future Biopharmaceutical Human Resources Training and Research Team, Seoul 133-791, Republic of Korea ‡

S Supporting Information *

ABSTRACT: Developing an artificial extracellular matrix that closely mimics the native tissue microenvironment is important for use as both a cell culture platform for controlling cell fate and an in vitro model system for investigating the role of the cellular microenvironment. Electrospinning, one of the methods for fabricating structures that mimic the native ECM, is a promising technique for creating fibrous platforms. It is well-known that align or randomly distributed electrospun fibers provide cellular contact guidance in a single pattern. However, native tissues have hierarchical structures, i.e., topographies on the micro- and nanoscales, rather than a single structure. Thus, we fabricated randomly distributed nanofibrous (720 ± 80 nm in diameter) platforms via a conventional electrospinning process, and then we generated microscale grooves using a femtosecond laser ablation process to develop engineered fibrous platforms with patterned hierarchical topographies. The engineered fibrous platforms can regulate cellular adhesive morphology, proliferation, and distinct distribution of focal adhesion proteins. Furthermore, confluent myoblasts cultured on the engineered fibrous platforms revealed that the direction of myotube assembly can be controlled. These results indicate that our engineered fibrous platforms may be useful tools in investigating the roles of nano- and microscale topographies in the communication between cells and ECM. KEYWORDS: ECM (extracellular matrix), hierarchical topographies, nano/micro scale, electrospinning, femtosecond laser

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fundamental functional unit of the muscle fiber that enables inherent muscle functions such as relaxation and contraction.7,8 Considerable effort has been devoted to fabricating bioinspired platforms using electrospinning techniques for engineering artificial tissues.9−11 Electrospinning processes have been developed to produce fibrous platforms that mimic the native ECM. Electrospinning can easily fabricate fibrous structures with various diameters on the nano- or micrometer scale. The fibrous platforms possess high contact surface areas that provide considerable space for cell attachment and utilize geometric patterns depending on the electrospinning parameters, such as polymer types, voltage, flow rate, and mandrel speeds.12 Moreover, it is possible to create anisotropically deposited fibrous platforms that could be used to encourage

INTRODUCTION Cells are surrounded by complex three-dimensional networks of extracellular matrix (ECM) that provides chemical and physical stimuli to determine the fate of cells.1,2 Cells isolated from their original cellular environments in the body are prone to losing their characteristics, particularly when differentiated in vitro. Therefore, engineered cell culture platforms are needed to provide environmental cues similar to those of the original ECM to manipulate cell fate in vitro and subsequently aid tissue function in vivo.3 Note that each type of tissue constituting the human body possesses unique structural characteristics that are prerequisites for its inherent function.4,5 In particular, skeletal muscle is a highly striated tissue and is found on peripheral bone in the form of collagen fibers known as tendons.6 During myogenesis, myoblasts fuse to form myofibers, which have characteristics of long, cylindrical, and multinucleated myotubes. Subsequently, repeated myofibers in a unit are called a sarcomere, which is the © 2016 American Chemical Society

Received: November 25, 2015 Accepted: January 15, 2016 Published: January 15, 2016 3407

DOI: 10.1021/acsami.5b11418 ACS Appl. Mater. Interfaces 2016, 8, 3407−3417

Research Article

ACS Applied Materials & Interfaces

microscale grooves. Our results demonstrated that engineered fibrous platforms with patterned hierarchical topographies can modulate myoblast adhesion, morphology, proliferation, and differentiation, thus suggesting that our engineered fibrous platforms may closely mimic the natural fibrous aspects of the ECM, which can control cell fate. Furthermore, it is applicable in vitro as a model system that can shed light on the regulatory mechanism in cell−ECM interactions.

myoblast differentiation to form multinucleated myofibers that have structural similarity between native muscle tissue and fabricated artificial muscle tissue.13,14 For example, Ku et al. reported that myoblasts cultured on aligned PLLA fibers using a high-speed mandrel presented a well-arranged morphology and improved formation of myotubes compared to cells cultured on random fibers.13 Huang et al. reported that stretching randomly distributed fibers to 200% deformation in length at 60 °C resulted in the fabrication of uniaxially aligned fibers that regulated cell alignment, myotube assembly, and myotube direction.14 However, one of the major concerns regarding conventional electrospinning methods is that densely deposited fibrous shapes with low porosity can hinder the efficient infiltration of cells, which may prevent complete cellular integration with the host tissue during in vivo transplantation.15−17 Moreover, previous reports on fibrous platforms have focused on creating unitary patterned fibrous platforms such as aligned fibers or random fibers on the nano- or microscale, which limits the complex structures of native tissues that they can mimic. To overcome these limitations, many attempts have been conducted using the following approaches: salt-leaching and cryogenic electrospinning to increase the pore size and layerby-layer electrospinning to form thick multilayered fibrous platforms consisting of nano- and microfibers.18 Despite these efforts, these approaches still result in mechanical deformation during transit depending on the artificially modified fibrous platforms or on the electrospinning method. Additionally, there have been reported that cellular adhesion interactions on align and random fibers exhibit different aspects from each other.19 Therefore, ideal engineered fibrous platforms should possess high porosity to provide better diffusion of nutrients and cell ingrowth and reasonable complex structures to imitate the complex structure of native tissue and preserve the mechanical properties. Recently, the laser processing of materials, known as ablation, has proven to be an invaluable technique for obtaining desired patterns on target materials through a single-step fabrication process.20 A femtosecond laser applies ultrashort laser pulses (