Enhancement of neurite outgrowth using nano-structured scaffolds coupled with laminin

Abstract

Cell interactions with scaffolds are important for cell and tissue development in the process of repairing and regeneration of damaged tissue. Scaffolds that mimic extracellular matrix (ECM) surface topography, mechanical stiffness, and chemical composition will be advantageous to promote enhanced cell interactions. Electrospinning can easily produce nano-structured synthetic polymer mats with architecture that structurally resembles the ECM of tissue. Although electrospinning can produce sub-micron fibrous scaffolds, modification of electrospun scaffolds with bioactive molecules is beneficial as this can create an environment that consists of biochemical cues to further promote cell adhesion, proliferation and differentiation. Incorporation of laminin, a neurite promoting ECM protein, onto the nanofibers is an alternative to further mimic the biochemical properties of the nervous tissue to create a biomimetic scaffold. In this study, we investigated the feasibility to functionalize scaffolds by coupling laminin onto poly(l-lactic acid) (PLLA) nanofibers. Laminin was successfully added to nanofibers using covalent binding, physical adsorption or blended electrospinning procedures. PC12 cell viability and neurite outgrowth assays confirmed that the functionalized nanofibers were able to enhance axonal extensions. Significantly, compared to covalent immobilization and physical adsorption, blended electrospinning of laminin and synthetic polymer is a facile and efficient method to modify nanofibers for the fabrication of a biomimetic scaffold. Using these functionalization techniques, nanofibers can be effectively modified with laminin for potential use in peripheral nerve regeneration applications.

Introduction

Enhancing cell interactions with tissue-engineered biomaterial is crucial for successful applications of scaffolds for repairing and regenerating damaged tissue. Scaffolds can be designed to mimic complex biological structures, or provide the mechanical support to allow the cells of the damaged tissue to remodel and repair to form distinct three-dimensional tissue structures that resemble the original tissue [1], [2]. In recent years, electrospinning has been extensively used to construct tissue-engineered scaffolds because it is a simple fabrication process that can easily produce nano- and micro-size synthetic polymeric fibers. Electrospun fibers are structurally analogous to the naturally occurring protein fibrils/fibers in the extracellular matrix (ECM) of the body [3], [4]. Briefly, this technique utilizes an electric field generated by an applied voltage that subsequently introduces surface charges on the polymer solution. This thus induces the formation of a Taylor cone polymeric droplet at the tip of the spinneret. As the electric potential that is created at the droplet surface exceeds a critical value, the electrostatic forces will overcome the solution surface tension to initiate polymer jet stream. Nanofibers can then be drawn from the polymer jet stream, and collected on the grounded collector as the solvent evaporates [3], [5]. Synthetic polymeric nanofibers such as poly(ɛ-caprolactone) [6], poly(l-lactic acid) [7], poly(glycolic acid) [8] and poly(dl-lactic-co-glycolic acid) [3], and naturally occurring polymeric nanofibers such as collagen [9] and gelatin [10] have been electrospun for studies in bone [6], vascular [11], peripheral nerve [7], [12], and other tissue-engineered grafts.

Poly(l-lactic acid) (PLLA) is a widely used material for scaffold fabrication because it is biodegradable and generally shows good biocompatibility. Also, PLLA possesses sufficient mechanical integrity to be used as the scaffold material for nerve guidance channels fabrication [13]. In neural tissue engineering, porous PLLA conduits [14], [15] and PLLA filaments [16], [17] were evaluated in rat sciatic nerve model that yielded promising results for applications in peripheral nerve regeneration. Nano-textured PLLA scaffolds made up of nano-fibrils have been shown to allow the adhesion and proliferation of neural cells [7]. However, PLLA does not have biological recognition sites that can interact with the cells. It is therefore desirable to modify PLLA nanofibers using simple methods to improve the biocompatibility of PLLA to enhance cell–matrix interaction. Also, PLLA is generally hydrophobic due to the non-polar groups along its backbone, and synthetic polymer nanofibers are relatively hydrophobic as the decrease in fiber diameter will correspond to an increase in effective contact angle [18]. Improving the hydrophilic property of electrospun and incorporation of cell-recognition domains such as RGD onto nanofibers can be done to enhance cell–scaffold interactions. In a previous study [19], air plasma treatment was used to improve the hydrophilic property of electrospun materials. In the same study, gelatin was grafted onto the nanofibers to promote cell spreading and proliferation. Nanofibers had also been modified using collagen coating technique [11] or blending collagen in polymer solution for electrospinning [5] to enhance cell attachment and viability. These studies showed that nanofibers can be easily modified with ECM bioactive proteins to enhance interactions of the scaffolds with cultured cells.

During development of the peripheral nervous systems (PNS), haptotactic factors are known to influence and stimulate axon guidance and neurite extension. Haptotactic cues include the incorporation of contact-mediated signals, such as ECM proteins or short sequences of the “functional” nucleotides of the ECM proteins, to guide axons to their synaptic targets [20]. In this study, laminin will be used as the contact guidance biochemical cues for axonal outgrowth. Laminin [21], [22] is one of the ECM component that is continuously synthesized after nerve injury [23] and it plays a crucial role in cell migration, differentiation and axonal growth [22], [24]. For example, myelination in the PNS is affected by laminin. Studies have described that even as Schwann cells could proliferate and migrate along axons, differentiation of myelinating phenotype was not observed without the presence of laminin [25], [26]. Furthermore, in vitro experiments have shown that neurite outgrowth is enhanced on scaffolds that were covalently bound with laminin [27]. Physical adsorption of laminin onto substrates with microgrooves [28] or microfilaments that acted as intra-luminal support matrices [17] have also been evaluated. These studies showed that in vitro directional guidance of the neurite outgrowth was achieved and enhanced using scaffolds that were physically adsorbed with laminin. Improved axonal outgrowth has also been observed in nerve guides filled with laminin gel as well [29]. Therefore, the incorporation of laminin onto nanofibers can potentially improve the rate of nerve regeneration.

In this study, the aim was to investigate and compare the chemical composition of modified PLLA nanofibers through the addition of laminin by covalently binding, physical adsorption, or blending with PLLA polymer solution during electrospinning. In addition, neural cell viability and differentiation were analyzed to determine the suitability of using laminin modified PLLA nanofibers and compare the efficiency of the modification methods for peripheral nerve regeneration applications.