Spatial coordination of cell orientation directed by nanoribbon sheets

Abstract

Spatial coordination of cell orientation is of central importance in tissue/organ construction. In this study, we developed microfabricated poly(lactic-co-glycolic acid) (PLGA) nanoribbon sheets with unique structures, using spin-coating and micropatterning techniques, in order to generate a hierarchically assembled cellular structure consisting of murine skeletal myoblasts (C2C12). The nanoribbon sheets were composed of aligned PLGA nanoribbons in the center, and strips on four sides which take a role as bridges to connect and immobilize the aligned nanoribbons. Such unique structures facilitated the alignment of C2C12 cells into bilayer cell sheets, and cellular alignment was directed by the aligned direction of nanoribbons. The nanoribbon sheets also facilitated the construction of multilayer cell sheets with anisotropic (orthogonal) and isotropic (parallel) orientations. The enhanced expression of myogenic genes of C2C12 cells on the bilayer cell sheets demonstrated that the nanoribbons induced C2C12 cell differentiation into mature myoblasts. The micropatterned nanoribbon sheets may be a useful tool for directing cellular organization with defined alignment for regenerative medicine and drug screening applications.

Introduction

Recapitulation of hierarchical assembly of cells and extracellular matrix (ECM) is of great significance to engineer functional tissues. The exquisite microscale and nanoscale architectures of tissues induce various cell behaviors, including alterations in cell adhesion, morphology, orientation, and various intracellular signaling pathways [1], [2], [3]. For example, cardiac and skeletal muscles possess striated structures consisting of sarcomeres packed into highly organized bundles. Skeletal muscle fibers are organized as parallel bundles, whereas cardiac muscle fibers are arranged at branching angles [4], [5]. To engineer an artificial niche or scaffold that simulates the natural topographic landscape of tissues (e.g. muscles), one potential approach is to construct micro-organized cellular structures. Efforts are underway to engineer multilayer cell-material composites using various techniques, including laser-guided directed writing, micro-molding of cell-laden hydrogels, and dielectrophoretic force-cell micropatterning [6], [7], [8]. However, these techniques have been limited by complicated process requirements and a lack of suitable biological properties. For example, alginate hydrogels have been widely used to develop microparticles and microfibers; however, anchorage-dependent cells poorly proliferate on these substrates.

Micropatterning, which facilitates the generation of simple or complicated motifs (e.g. grooves, pillars, and wells) on various surfaces with flat or curved features, is a powerful tool for directing cell behaviors, including spatial arrangement and differentiation [9], [10], [11]. However, despite the significant efforts, the construction of a three-dimensional (3D) structure with a well-defined cell arrangement using micropatterning remains a challenge [12]. To address this challenge, we aimed to develop a technique that combines the superior qualities of micropatterning to build multilayered cellular constructs.

Advances in nanotechnology have led to the development of freestanding, ultrathin polymeric films (referred to as “nanosheets”). Nanosheets, which are composed of biodegradable and biocompatible materials (e.g. polylactic acid and polysaccharides), have unique features, such as strong adhesion to dry or hydrated surfaces, high flexibility (allowing adherence to flat or curved surfaces), and excellent transparency [13], [14], [15], [16], [17]. Here, we aimed to utilize these properties to develop engineered matrices for constructing multilayer micropatterned cell sheets. To this end, it was important for the nanosheets to display excellent biodegradability and cell adhesion as well as low cytotoxicity to facilitate cell residence and the regulation of cell orientation [18]. Poly(lactic-co-glycolic acid) (PLGA) is a copolymer approved by the Food and Drug Administration (FDA) for use in a number of therapeutic devices [19], [20]. The tunable degradation (regulated by the composition of lactic and glycolic acids) and safety of PLGA (commercial products include Bone-Fix and drug-releasing microspheres) have been confirmed [21].

In this study, we developed microfabricated PLGA nanoribbon sheets with a unique structure using spin-coating and micropatterning techniques to generate a hierarchically assembled cellular structure consisting of aligned murine skeletal myoblasts (C2C12). This material bridges the gap between the well-developed two-dimensional (2D) micropatterning for cell patterning and the 3D construction of multicellular structures. We anticipate that the nanoribbons may be a useful tissue engineering tool for directing cellular organization with defined alignment and microstructure, which could be useful for engineering 3D tissue structures for applications in regenerative medicine and drug screening. For example, freestanding aligned cell sheets could serve as a multilayered cardiac patch, artificial skin, or engineered skeletal muscle.