| Peripheral nerve injury is one of the most common clinical traumatic diseases. Peripheral nerve injury with a large gap often leads to lack of movement and sensory function. It has a high disability rate and serious impact on the quality of life. Although peripheral nerves can regenerate after injury, the spontaneous nerve regeneration is usually incomplete and the functional recovery is poor. Therefore, clinical treatments are needed to promote nerve repair. At present, the gold standard of peripheral nerve repair is the autologous nerve graft. But it is limited by a limited source of nerve, the damage of the donor site and mismatching between the donor nerve and the injured nerve. So there is a need to find new ways to improve peripheral nerve regeneration. Nerve guidance conduits (NGCs) can provide structural guidance and microenvironment support for nerve regeneration. At present, a variety of synthetic materials and natural materials have been used in the preparation of nerve conduits. The repair effect of some nerve conduits are close to that of the autologous nerve graft. The existing nerve conduits need further study. At the same time, it is necessary to develop new biomaterials for peripheral nerve repair.Cellulose is one of the most abundant natural polymers, which has good biocompatibility and mechanical properties. Soy protein isolate (SPI) has good biodegradability, biocompatibility and processability, which has been used in wound dressings, bone tissue engineering and drug controlled release. In our previous work, we successfully modified cellulose using SPI. The cellulose/SPI based films and sponges showed good biocompatibility in vitro and in vivo. It has been confirmed that NGCs fabricated from cellulose/SPI based films (CSFC) promoted nerve regeneration in a rat model with a 10 mm long sciatic nerve gap. However,3 months after the surgery, CSFC had not degraded, and recovery of nerve function was not satisfactory. Therefore, it is needed to optimize cellulose/SPI composite nerve conduit and improve the biodegradability and nerve repair efficiency.We have previously demonstrated that porous cellulose/SPI composite sponge can be prepared using a freeze-drying process. The cellulose/SPI composite sponge had good permeability, biocompatibility and biodegradability. Therefore, cellulose/SPI composite sponge can be used to optimize CSFC.In addition to the ultrastructure of the materials, bioactive factors and the structure parameters of nerve conduits also have important influences on nerve repair efficiency. Pyrroloquinoline quinine (PQQ) is a small bioactive substance. It can promote the formation of endogenous NGF and promote nerve regeneration. Therefore, the addition of PQQ may be conducive to nerve regeneration. Structure parameters of nerve conduits such as the diameters and the numbers of channels also have impacts on nerve regeneration. For example, multichannel nerve conduits simulate the normal structure of nerves and can reduce the dispersion of axonal regeneration. So, multichannel nerve conduits are beneficial to nerve regeneration and functional recovery. Therefore, preparation of single channel nerve conduits with different channel diameters and multichannel nerve conduits can be used as another two ways to optimize cellulose/SPI sponge-based conduits.Therefore, in this paper, cellulose and soy protein isolate were used to prepare cellulose/SPI film based conduit and cellulose/SPI sponge-based conduit (CSSC) with the same chemical components and different physical structures. Effects of CSSC and CSFC on regeneration of the defective nerve were comparatively investigated in rats with a 10 mm long gap in sciatic nerve. The probable molecular mechanism was also investigated. On the basis of this work, we optimized CSSC from the two aspects: firstly, we introduced bioactive PQQ into CSSC and evaluated the repair effects in vivo. Secondly, we optimized the structure parameters of CSSCs. We prepared single channel CSSCs with different inner diameters and multi-channel CSSCs, then evaluated the repair effects of these nerve conduits on peripheral nerve defects. The main contents of this work include the following aspects:(1) Single channel CSSCs were prepared. The morphology, porosity, water absorption and in vitro degradation were evaluated. The results showed that single channel CSSCs had porous structure. CSSCs had higher porosity and water absorption, suggesting that the permeability was good. In addition, CSSCs also had better in vitro degradation. It showed that the cellulose/SPI sponge had the potential to be used as a nerve conduit.(2)The repair efficiency of single channel CSFC and CSSC was comparatively evaluated by a combination of electrophysiological assessment, Fluoro-Gold retrograde tracing, double NF200/S100 immunofluorescence analysis, toluidine blue staining, and electron microscopy. The probable molecular mechanism was investigated using quantitative real-time PCR (qPCR) analysis. The results showed that the repair efficiency of CSSC was higher than that of CSFC, which may be attributed to a porous structure and a more favourable microenvironment for nerve regeneration in CSSC.(3)The impact of PQQ on the repair efficiency of CSSC was investigated in rats. The structure and function recovery of regenerated nerves in CSSC-PQQ and CSSC were evaluated to observe the effects of PQQ on nerve repair. The results showed that compared with the pure CSSC nerve conduit, the CSSC-PQQ composite nerve conduit had better repair efficiency with better nerve structural and functional recovery. It was showed that PQQ had the function of promoting nerve repair.(4)The impact of channel diameter on the repair efficiency of single channel CSSC was observed in vivo. Single channel CSSCs with different channel diameters were prepared and used to bridge a 10 mm long sciatic nerve defect in rats. The structural and functional recovery of regenerated nerves was evaluated to observe the influence of channel diameter on nerve repair. It was found that the repair efficiency of nerve conduits with inner diameters of 1.91 mm and 2.31 mm was better, while the repair efficiency of nerve conduit with an inner diameter of 1.27 mm was poor. The results showed that the repair effect was better when the inner diameter of the nerve conduit was slightly larger than the diameter of the injured nerve.(5)The impact of channel number on the repair efficiency of CSSC was studied. Multi-channel and single channel CSSCs were prepared and used to repair a 10 mm long sciatic nerve defect in rats. The structural and functional recovery of regenerated nerves was evaluated to observe the impact of channel number on nerve repair. The results showed that the structure and functional recovery of regenerated nerves in 3-channel CSSC group was better than those in single channel CSSC group and 7-channel CSSC group. It suggested that the 3-channel structure might be the best for CSSC. The possible mechanisms may be that 3-channel CSSC can reduce the dispersion of axonal regeneration.3-channel CSSC also had a larger inner surface area and was conducive to cell adhesion and growth.In summary, CSSCs were prepared and the morphology, porosity, water absorption and in vitro degradation were detected. The repair efficiency of CSSC was evaluated in a rat sciatic nerve defect model. CSSC was optimized by introducing PQQ, changing the inner diameter and the number of channels. It was proved that CSSC can be used for nerve repair, which can be optimized through a variety of ways. CSSC had good application prospects in the field of peripheral nerve tissue engineering. |
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