A synthetic oxygen carrier in fibrin matrices promotes sciatic nerve regeneration in rats.

Tissue-engineering nerve conduits have been studied for a long time in bridging large nerve defects. However, the low oxygen availability within the nerve conduits, which results in death of migratory Schwann cells (SC) or loss of the newly formed tissue's function, is still an obstacle for axonal regeneration. Thus, it was hypothesized that an oxygen-enriched conduit would enhance axonal regeneration and functional recovery in vivo. To address this issue, perfluorotributylamine (PFTBA) enriched fibrin hydrogel was prepared and injected into collagen-chitosan conduits. The conduit containing PFTBA-enriched fibrin hydrogel was then used to bridge a 12-mm sciatic nerve defect in rats. The control rats were bridged with collagen-chitosan conduits filled with fibrin matrices without PFTBA. It was found that axonal regeneration and functional recovery in the combined PFTBA group were significantly higher than those in the control group without PFTBA. Further investigations showed that the mRNA and protein levels of S-100, brain-derived neurotrophic factor and nerve growth factor were enhanced by PFTBA at 1 and 3weeks after surgery. However, the mRNA and protein levels of vascular endothelial growth factor were in a similar range between the combined PFTBA group and the control group without PFTBA. In addition, immunohistochemical results showed that the morphological appearances of regenerated nerve and survival of SC were enhanced by PFTBA at 4 and 12weeks after surgery. In conclusion, PFTBA-enriched nerve conduit is capable of enhancing axonal regeneration, which provides a new avenue for achieving better functional recovery in the treatment of nerve defect.

Wang Y ，Qi F ，Zhu S ，Ye Z ，Ma T ，Hu X ，Huang J ，Luo Z ... -  《-》

Enhanced in vivo survival of Schwann cells by a synthetic oxygen carrier promotes sciatic nerve regeneration and functional recovery.

Local hypoxia in the early stages of peripheral nerve injury is a challenge for axonal regeneration. To address this issue, perfluorotributylamine (PFTBA)-based oxygen carrying fibrin hydrogel was prepared and injected into Schwann cell (SC)-seeded collagen-chitosan conduits to increase oxygen supply to SCs within the conduits. The conduit containing PFTBA-SC gel was then applied to bridge a 15-mm sciatic nerve defect in rats. It was observed that most of the GFP-labeled SCs initially seeded in the PFTBA hydrogel remained alive for approximately 28 days after their in vivo implantation. The number of SCs was significantly higher in the PFTBA-SC scaffold than that in the SC scaffold without PFTBA. In addition, nerve regeneration and functional recovery were examined after nerve injury repair. We found that the PFTBA-SC scaffold was capable of promoting axonal regeneration and remyelination of the regenerated axons. Further studies showed the PFTBA-SC scaffold was able to accelerate the recovery of motor and sensory function of the regenerating nerves. Electrophysiological analysis showed area under the curve of compound muscle action potential and nerve conduction velocity were also improved, and gastrocnemius muscle atrophy was partially reversed by PFTBA-SC scaffold. Furthermore, microvessel density analysis showed PFTBA-SC composites were beneficial for microvascular growth, which provided sustained oxygen for regenerating nerve in the later stages of nerve regeneration. In conclusion, enhanced survival of SCs by PFTBA is capable of promoting sciatic nerve regeneration and functional recovery, which provides a new avenue for achieving better functional recovery in the treatment of peripheral nerve injuries. Copyright © 2016 John Wiley & Sons, Ltd.

Ma T ，Zhu L ，Yang Y ，Quan X ，Huang L ，Liu Z ，Sun Z ，Zhu S ，Huang J ，Luo Z ... -  《-》

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

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.

Ma T ，Wang Y ，Qi F ，Zhu S ，Huang L ，Liu Z ，Huang J ，Luo Z ... -  《-》

A synthetic oxygen carrier-olfactory ensheathing cell composition system for the promotion of sciatic nerve regeneration.

The treatment of lengthy peripheral nerve defects is challenging in the field of the regenerative medicine. Thus far, many nerve scaffolds with seeded cells have been developed, which hold great potential to replace nerve autograft in bridging lengthy nerve defects by providing guiding and bioactive cues. However, low oxygen status has been found within nerve scaffolds after their implantation in vivo, which has been shown to result in death or loss of function of supportive cells, and significantly limit nerve regeneration and functional recovery after nerve injury. In the present study, perfluorotributylamine (PFTBA) was introduced into a collagen-chitosan conduit within which olfactory ensheathing cells (OECs) were seeded to increase oxygen supply to OECs, as well as regenerating axons. The "PFTBA-OECs" enriched scaffolds were then used to bridge a 15-mm-long sciatic nerve defect in rats. Both nerve regeneration and functional recovery were examined at pre-defined time points after surgery. We found that the number of GFP-labeled OECs was significantly higher in the "PFTBA-OECs" scaffold than that in the single OECs scaffold. In addition, PFTBA was found to enhance the beneficial effect of OECs-enriched scaffold on axonal regeneration and functional recovery. All these findings indicate that the "PFTBA-OECs" enriched scaffolds are capable of promoting nerve regeneration and functional recovery, which might be attributable, at least in part, to their beneficial effect on the survival of OECs after their implantation in vivo.

Zhu S ，Ge J ，Wang Y ，Qi F ，Ma T ，Wang M ，Yang Y ，Liu Z ，Huang J ，Luo Z ... -  《-》

Oxygen carrier in core-shell fibers synthesized by coaxial electrospinning enhances Schwann cell survival and nerve regeneration.

Rationale: Local hypoxia is a challenge for fabrication of cellular grafts and treatment of peripheral nerve injury. In our previous studies, we demonstrated that perfluorotributylamine (PFTBA) could provide short term oxygen supply to Schwann cells (SCs) and counteract the detrimental effects of hypoxia on SCs during the early stages of nerve injury. However, the quick release of oxygen in PFTBA compromised its ability to counteract hypoxia over an extended time, limiting its performance in peripheral nerve injury. Methods: In this study, PFTBA-based oxygen carrier systems were prepared through coaxial electrospinning to prolong the time course of oxygen release. Core-shell structures were fabricated, optimized, and the oxygen kinetics of PFTBA-enriched core-shell fibers evaluated. The effect of core-shells on the survival and function of SCs was examined in both 2D and 3D systems as well as in vivo. The system was used to bridge large sciatic nerve defects in rats. Results: PFTBA core-shell fibers provided high levels of oxygen to SCs in vitro, enhancing their survival, and increasing NGF, BDNF, and VEGF expression in 2D and 3D culture systems under hypoxic condition. In vivo analysis showed that the majority of GFP-expressing SCs in the PFTBA conduit remained viable 14 days post-implantation. We found that axons in PFTBA oxygen carrier scaffold improved axonal regeneration, remyelination, and recovery. Conclusion: A synthetic oxygen carrier in core-shell fibers was fabricated by the coaxial electrospinning technique and was capable of enhancing SC survival and nerve regeneration by prolonged oxygen supply. These findings provide a new strategy for fabricating cellular scaffolds to achieve regeneration in peripheral nerve injury treatment and other aerobic tissue injuries.

Ma T ，Yang Y ，Quan X ，Lu L ，Xia B ，Gao J ，Qi F ，Li S ，Zhao L ，Mei L ，Zheng Y ，Shen Y ，Luo Z ，Jin Y ，Huang J ... -  《Theranostics》