Bridging peripheral nerve defects with a tissue engineered nerve graft composed of an in vitro cultured nerve equivalent and a silk fibroin-based scaffold

Abstract

Tissue engineered nerve grafts are considered as a promising alternative to autologous nerve grafts used for peripheral nerve repair. The differences between these two types of nerve grafts are mainly in the regenerative microenvironment established by them. To construct ideal tissue engineered nerve grafts, it is therefore required to develop a better way to introduce biochemical cues into a neural scaffold, as compared to single or combined use of support cells and growth factors. Here, we used a co-culture system of dorsal root ganglia and Schwann cells to create an in vitro formed nerve equivalent, which was introduced into a silk fibroin-based scaffold to furnish a tissue engineered nerve graft (TENG). At 4- and 12- weeks after the TENG was implanted to bridge a 10-mm-long sciatic nerve defect in rats, histological and functional assessments as well as Western blot analysis were performed to evaluate the influences of the TENG on peripheral nerve regeneration. We found that at an early stage of nerve regeneration, the TENG significantly accelerated axonal growth, and up-regulated expressions of N-cadherin and PMP22. Twelve weeks after nerve grafting, the TENG produced a further improved outcome of nerve regeneration and functional recovery, which was more close to that of the autologous nerve graft than that of the silk fibroin-based scaffold. The introduction of an in vitro cultured nerve equivalent into a scaffold might contribute to establishing a native-like microenvironment for nerve regeneration.

Introduction

Peripheral nerve defects are clinically treated with implantation of an autologous nerve graft, which by far offers the best outcome of nerve regeneration and function restoration. This recognized “gold standard” strategy of peripheral nerve repair, however, suffers from several drawbacks, and the development of an alternative to autologous nerve grafts motivates great endeavors of neuroscientists and surgeons [1]. Now, tissue engineered nerve grafts provide an ideal option among various current alternatives. They are typically composed of a physical scaffold and biochemical cues. The former is usually prepared with a wide range of biomaterials, mainly exerting the actions of contact guidance and diffusion barrier for axonal regrowth. The latter is furnished by support cells and/or growth factors, mainly aiding to create a favorable microenvironment for peripheral nerve regeneration [2], [3], [4].

Schwann cells (SCs) are the main glial cells in the peripheral nervous system (PNS) and play an indispensable role in peripheral nerve regeneration. They become the first and most widely used support cells for introduction to the scaffold of tissue engineered nerve grafts, thus improving nerve regeneration in experimental studies [5], [6], [7]. To expand the source of support cells, stem cells, especially bone marrow mesenchymal stem cells, are tried to serve as support cells within tissue engineered nerve grafts with considerable success in peripheral nerve repair [8], [9], [10], [11]. On the other hand, the use of growth factors and other bioactive molecules for tissue engineered nerve grafts has also been demonstrated to exert positive effects on peripheral nerve regeneration [12], [13].

It seems that the differences between tissue engineered nerve grafts and autologous nerve grafts are mainly in the regenerative microenvironment established by them. To overcome the congenital deficiency of tissue engineered nerve grafts and create an optimal microenvironment for peripheral nerve regeneration, we attempted to seek a better way, as compared to single or combined use of support cells and growth factors, to introduce biochemical cues into a scaffold. Here we tested a hypothesis whether a co-culture system of dorsal root ganglia (DRGs) and SCs could give rise to an in vitro cultured nerve equivalent that was likely to mimic the native nerve microenvironment, and we further integrated these nerve-like tissues with the silk fibroin (SF)-based scaffold to engineer a nerve graft, which was implanted to bridge a 10-mm defect in the rat sciatic nerve. The process of nerve regeneration was monitored at an early stage of 4 weeks after grafting, and the regenerative outcomes were evaluated at 12 weeks after grafting by functional, morphological and histological assessments.