The effect of synthetic oxygen carrier-enriched fibrin hydrogel on Schwann cells under hypoxia condition in vitro

Abstract

Schwann cell (SC), which plays a key role in peripheral nerve regeneration, is one of the most classic supportive cells in neural tissue engineering. However, the biological activity of SCs seeded in nerve scaffolds decays subsequently due to local hypoxia induced by ischemia. Thus, we aimed to investigate whether a synthetic oxygen carrier-enriched fibrin gel would provide a sustained oxygen release to cultured SCs in vitro for overcoming a temporary (48 h) oxygen deprivation. In this study, perfluorotributylamine (PFTBA)-based oxygen carrying fibrin gel was prepared to provide oxygen for SCs under normoxic or hypoxic conditions. The dissolved oxygen within the culture media was measured by a blood-gas analyzer to quantify the time course of oxygen release from the PFTBA-enriched fibrin gel. SCs were cultured in the presence or absence of PFTBA-enriched fibrin gel under normoxic or hypoxic conditions. The tolerance of SCs to hypoxia was examined by a cell apoptosis assay. The growth of cells was characterized using S-100 staining and a CCK-8 assay. The migration of cells was examined using a Transwell chamber. The mRNA of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), glial cell derived neurotrophic factor (GDNF), neural cell adhesion molecule (N-CAM) and vascular endothelial growth factor (VEGF) in SCs were assayed by RT-PCR. In addition, SCs cultured in 3D PFTBA-enriched hydrogel were characterized by Live/Dead staining and the mRNA levels of BDNF, NGF, GDNF, N-CAM and VEGF were assayed by RT-PCR. The results showed that the PFTBA-enriched fibrin hydrogel was able to promote cell adhesion, migration, and proliferation under hypoxic conditions. Interestingly, PFTBA applied through the fibrin hydrogel dramatically enhanced the mRNA of BDNF, NGF, GDNF, N-CAM and VEGF under hypoxic condition. These findings highlight the possibility of enhancing nerve regeneration in cellular nerve grafts through PFTBA increased neurotropic secretion in SCs.

Introduction

Schwann cells, as one of the most classic supportive cells in neural tissue engineering, have been proven to be able to provide bioactive substrates for axonal migration and release molecules that regulate axonal outgrowth [1]. In addition, they are the myelin-forming cells of the peripheral nervous system, which play a key role in peripheral nerve regeneration. Driven by the above considerations, several studies introduced SCs into nerve scaffolds in order to improve nerve regeneration and functional recovery [2], [3], [4]. However, more and more studies have shown that the biological activity of SCs seeded in nerve scaffolds decayed subsequently due to local hypoxia condition, which significantly limited their performance in supporting nerve regeneration within nerve scaffold [5], [6]. Therefore, a sustained oxygen supply to SCs holds the potential to further improve the performance of cell-based nerve grafts in promoting nerve regeneration and functional recovery in the repair of lengthy nerve defects.

In case of lengthy nerve defects, the blood supply was severely damaged, or even absent at the local site of nerve defect. When cell-based nerve grafts were used to bridge the two dissociative stumps, the cells within nerve scaffolds were exposed to temporary hypoxia despite of oxygen diffusion from surrounding tissues [7]. Many studies have found that most of cells die at the early stage of transplantation in vivo, for which hypoxia is one of the major causes [8], [9], [10]. Thus far, several strategies have been employed to improve oxygen diffusion or supply by controlling pore size and porosity of nerve scaffolds, which always result in inefficient oxygen supply at the injured sites [11]. In addition, incorporating vasotropic factors into nerve scaffolds has also been attempted to provide oxygen for SCs by accelerating vascularization within nerve scaffold [12], [13]. However, it at least takes several days or even weeks to complete the process of vascularization within nerve scaffolds, which is unable to provide oxygen for SCs at the early stage of their transplantation.

To provide instant oxygen for SCs at the early stage of transplantation in vivo, one option is to increase the level of oxygen within the nerve scaffolds. It has been reported that PFTBA, one representative agent of perfluorocarbons (PFCs), has a high solubility of oxygen, which is 15–20 fold than that in water and 2–3 fold than that in blood [14]. It has been shown PFTBA emulsions have a linear relationship between oxygen partial pressure and oxygen concentration [15], [16]. In addition, PFTBA is advantageous because of its commercial availability, chemical and biological inertness, and ability to be easily sterilized [14]. All these properties make PFTBA an attractive addition to nerve graft to provide oxygen for SCs at the early stage of transplantation. Thus far, PFTBA has been reported to increase the oxygen level in bone tissue engineering scaffold and affect survival and differentiation of mesenchymal stem cells (MSCs) [17]. In addition, PFCs has also been shown to increase metabolic activity and survival in HepG2 liver cells [18]. Therefore, PFTBA might be able to further improve the performance of cell-based nerve grafts in promoting nerve regeneration by providing instant oxygen to cells within the nerve graft. Before this attempt, we have to clarify the effect of PFTBA on SCs under hypoxic conditions. Therefore, the present study was designed to investigate whether PFTBA would provide a sustained oxygen release to cultured SCs in vitro for overcoming a temporary oxygen deprivation, and further investigate the effect of PFTBA on SCs under hypoxic conditions.