Patterned growth and differentiation of neural cells on polymer derived carbon substrates with micro/nano structures in vitro

Abstract

The growth of neuroblastoma (N2a) and Schwann cells has been explored on polymer derived carbon substrates of varying micro and nanoscale geometries: resorcinol–formaldehyde (RF) gel derived carbon films and electrospun nanofibrous (∼200 nm diameter) mat and SU-8 (a negative photoresist) derived carbon micro-patterns. MTT assay and complementary lactate dehydrogenase (LDH) assay established cytocompatibility of RF derived carbon films and fibers over a period of 6 days in culture. The role of length scale of surface patterns in eliciting lineage-specific adaptive response along, across and on the interspacing between adjacent micropatterns (i.e., “on”, “across” and “off”) has been assayed. Textural features were found to affect 3′,5′-cyclic AMP sodium salt-induced neurite outgrowth, over a wide range of length scales: from ∼200 nm (carbon fibers) to ∼60 μm (carbon patterns). Despite their innate randomness, carbon nanofibers promoted preferential differentiation of N2a cells into neuronal lineage, similar to ordered micro-patterns. Our results, for the first time, conclusively demonstrate the potential of RF-gel and SU-8 derived carbon substrates as nerve tissue engineering platforms for guided proliferation and differentiation of neural cells in vitro.

Introduction

Neural dysfunctions, stemming from neurodegeneration or injury to the neural pathway of the central (CNS) or peripheral nervous system (PNS), are widespread [1]. Major neural damage may engender loss of memory, impaired sensory or motor function, muscle dysfunctions and neuropathic pains [2]. The complexity of neural tissue in terms of anatomy, functional structuring and information processing deters the application of current technologies subsuming nerve autografts [2], [3], allografts [4], xenografts [4], acellular grafts [5] and pre-degenerated nerve grafts [6]. This calls for the exploration of various biomaterials as better or effective neural tissue engineering platforms. For example, a polymer scaffold can be leveraged not only to promote neural cell adhesion [7], [8], [9], but also as a template to co-ordinate cell growth/differentiation and direct axonal growth to achieve real functional neuronal network [10], [11], [12], [13].

Since its first use in the 1960s [14], there has been burgeoning interest in carbon materials for biomedical applications (e.g., bio-microelectromechanical systems; bio-MEMs, biosensors) [15], particularly in orthopedics and neural tissue engineering [16]. Such widespread interest is due to their attractive electrochemical and physical properties and the ease of functionalization and porosity control [17], [18]. Also, a conductive scaffold itself can influence cell responses, like enhanced neural differentiation, neural extension and axon regeneration prior to tissue development, both in the presence or absence of an applied electric field [19].

Among carbon materials, carbon nanotubes (CNTs) stand out as well-suited platforms for neural tissue engineering, as their efficacy in promoting not only neuronal attachment, growth and differentiation, but also synaptic activity and network connectivity is being reported [20], [21]. Several pioneering reviews on the applicability of CNTs at the neural interfaces have been well documented [22], [23], [24]. Also, carbon nanofibers (CNFs) have been widely investigated for applications in regenerative medicine [25], [26]. CNFs can be fabricated by pyrolysis of suitable fiber-reinforced polymer precursors [27], [28], [29] and can be functionalized with biomolecules to form scaffolds for hosting neuron hybrids [30]. Zhou et al. [31] demonstrated longer and more abundant neurites per cell [rat neuroblastoma, carcinoma (P-19) and rat pheochromocytoma (PC-12)] on pyrolyzed carbon films than on control samples. At a larger length scale, micropatterned carbon substrates have been successfully harnessed as tissue engineering platforms [32], [33], [34]. Photolithography of polymer photoresists, followed by pyrolysis in an inert atmosphere, warrants an easy and robust method for fabrication of textured carbon surfaces [31], [35], [36].

Although biocompatibility of such carbon-MEMS has been studied by several research groups [37], [38], [39], the biocompatibility of carbon nanomaterials is constantly being debated [40], [41]. Additionally, the studies on polymer-derived carbon substrates in the context of neural tissue engineering applications are not yet reported comprehensively. More specifically, the compatibility of fibrous carbon substrates derived from resorcinol–formaldehyde (RF)-gel is not yet established. This served as the major motivation for the current work.

In this study, textured carbon substrates were engineered for investigation of neural cell behavior and compatibility. Effects of CNFs, films and micro-patterns on neural cell morphology, adhesion, viability, proliferation, differentiation and alignment were studied. RF precursor material was used to fabricate flat films and electrospun nanofibrous mats, which were subsequently carbonized in an inert atmosphere. RF gel is a widely known precursor for glassy carbon [42], [43]. Although RF gel has been harnessed for fabrication of carbon nanospheres [44] and patterned surfaces [45]; to the best of our knowledge, fabrication of electrospun RF-derived carbon fibers have not been reported previously. A study on patterned growth of neuroblastoma and Schwann cells on carbon substrates of various topographies has not been made previously. Hence, micropatterned carbon substrates were fabricated by photolithography on a widely used negative photoresist, SU-8, followed by pyrolysis [46]. Two different cell lines, mouse neuroblastoma (N2a) and Schwann cells were investigated keeping in mind their synergistic involvement in generation of axons, ability to differentiate into neurons [47] and to form myelin sheath for peripheral nerve regeneration. In fact, Schwann cells and neuronal cells exhibit reciprocal supportive interactions, which are central to the development of the PNS. The response of these neural cell lines to substrates with varying roughness, particularly, patterns of various dimensions and shapes in terms of morphological adaptability, viability and cell differentiation has been explored. It can be envisioned that a clear understanding of cell/substrate interactions in terms of the specific topographic cues furnished by the substrate will cater better conceptualization of the mechanisms involved in nerve repair and regeneration, the so-called “holy grail” of nerve tissue engineering.