| Objective: To assess the toxicity and biocompatibility of a microtube array sheet(MTAS) with a unique longitudinal surface topography,and study the potential of MTAS to promote peripheral nerve regeneration efficiency, both in vivo and in vitro.Methods: According to international standard ISO10993-5 toxic material detection methods,the preconditioned medium was prepared by incubating the electrospunning MTAS with DMEM medium for 24 h. To assess the toxicity and biocompatibility of MTAS, primary rat Schwann cells were cultured following preconditioned medium.And then MTT assay, Annexin V-PI staining was carried with treated cells to detect cell viability and apoptosis.Biological properties of Schwann cells, including proliferation, migration and neurotrophic factor production, which are critical in peripheral nerve regeneration, were evaluated with Brd-U test, transwell assay and Realtime-PCR test. And then Schwann cells, spinal cord motor neurons and dorsal root ganglion neurons were seeded on the MTAS to study the effect of the construct on the biological properties and behaviors of neural cells. Next, we tested the capability of MTAS to facilitate peripheral nerve regeneration by bridging a 10 mm sciatic nerve defect in rats with a nerve conduit equipped with MTAS lining. The effect of nerve regeneration was evaluated from morphology to function by immunofluorescence, toluidine blue staining, transmission electron microscopy, CMAP test, thermalgesia analysis and SFI analysis 8 w aftersurgery. And on this basis of the MTAS equipped nerve conduit,we further builded composite nerve conduits(Conduit+MTAS+SCs) by seeding primary SCs on MTAS as support cell, and use it to bridge a 10 mm sciatic nerve defect in rats, the efficiency of nerve regeneration was detected from morphology to function.Results: 1.The MTAS comprised assemblies of microtubes. Microtubes were aligned in parallel into a paper-thin sheet with a thickness of 50 μm. Numerous parallel and longitudinally oriented grooves were formed between two adjacent microtubes, constituting a unique surface geometry. Each groove had a V shape on the transverse section. The average depth of the groove was 8 μm, and the average width 20 μm. MTT data showed that the quantities of Schwann cells in both groups increased from 24 h and reached a plateau by 48 h. No significant differences were evident at each time-point, and treatment with preconditioned culture medium did not result in a left or right shift of the curve, suggesting that cell growth is not affected by preconditioned medium. Flow cytometry was further applied to examine the rate of cell apoptosis after a 2 day culture in preconditioned medium.Exposure to preconditioned medium did not lead to a significant increase in the apoptotic rate of cells. As indicated with Brd U analysis, no significant differences were observed between the control and preconditioned medium groups after a 2 day treatment period. Similarly, transwell analysis for cell migration capacity revealed no significant differences between the two groups. Real-time PCR analysis was applied to assess the capability of Schwann cells to express critical neurotrophic factors when cultured in preconditioned medium. The expression patterns of BDNF, CNTF, and NT4 remained similar at 24 and 48 h after treatment Slight but statistically significant increases in NGF and GDNF expression were recorded in the preconditioned medium group at 48 h. No significant differences were observed between the control and preconditioned medium groups in biological properties of Schwann cells, including proliferation, migration and neurotrophic factor production.2. To determine the effect of MTAS on behavioral changes of neural cells, primary cultured Schwann cells, DRG neurons and spinal cord motor neurons were cultured directly on the MTAS. Different from distributed at large in a spiral manner on petri dish,SCs are rearranged in a linear manner parallel to the grooves on the surface of the MTAS after 24 h culture, despite the initial random seeding. The cells in this group exhibited healthy bipolar morphology, extending long processes and connecting with each other from both ends.Similar patterns of neurite extension were observed in DRG neuron and spinal cord motor neuron cultures. 3.We employed 12 mm SF/P(LLA-CL) nerve conduits with MTAS membranous lining to bridge 10 mm sciatic nerve defects in rats with the aim of determining the efficiency of MTAS in promoting peripheral nerve regeneration in vivo. The injured nerve was found to regenerate into the nerve conduit and distal nerve stump by week 8 after repair in both the conduit-only and conduit plus MTAS groups.No significant differences were evident between the two groups with regard to the mean area of neural tissue, total number of myelinated fibers or myelin thickness in the mid-conduit section. However, the area of neural tissue in the distal nerve stump and the mean quantities of fibers in the conduit plus MTAS group were significantly larger than those in the conduit-only group.An obvious right shift in fiber caliber and left shift in G ratio was noted in the conduit plus MTAS group compared with that of the conduit-only group. Myelinated fibers exhibited completely different morphologies in the two groups. Nerve fibers in the conduit plus MTAS group presented a more rounded shape with an eccentricity value of 0.56±0.02, closer to that of the normal nerve(0.59±0.01), but were significantly smaller than that of the conduit group(0.83±0.02, p < 0.05), which displayed an oval and longitudinal shape, indicative of two different patterns of nerve growth. The muscle wet weight, average myocyte cross-sectional area, innervated degree of muscle and skin, CMAP peak and sciatic nerve function index had significant increases in conduit plus MTAS group(p<0.05). MTAS promoted regeneration ofsciatic nerve defect injury. 4.We further built composite nerve conduits(Conduit+MTAS+SCs) by seeding primary SCs on MTAS as support cell, and use it to bridge a 10 mm sciatic nerve defect in rats, the efficiency of nerve regeneration and the function recovery was significant improved compared with conduit plus MTAS group(p<0.05).Conclusions: MTAS with surface geometry of parallel and longitudinally aligned grooves successfully modified the biological behaviors of Schwann cells and peripheral neurons by guiding orientated cell alignment, migration and neurite extension from peripheral neurons in vitro without affecting the critical biological properties of Schwann cells, such as proliferation and neurotrophic factor expression, which are essential for constructing the regeneration microenvironment. When used as an inner lining of the nerve conduit, MTAS promoted regeneration, as indicated by the larger fiber caliber in the mid-conduit and greater quantity of fibers in the distal nerve stump. More importantly, the surface geometry guided orientated nerve growth along the MTAS toward the target, an essential requirement for efficient regeneration. Our collective in vivo and in vitro findings suggest that the novel MTAS is safe and effective in guiding orientated nerve growth without affecting the critical biological properties of Schwann cells. |
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