RESUMO
Objective To observe the effect of novel tissue engineered nerve grafts, which combined silk fi-broin/collagen scaffold with a co-culture of Schwann cells(SCs)and adipose-derived stem cells(ADSCs), to repair rat sciatic nerve defects. Methods From February, 2015 to August, 2016, the culture and purify SCs and ADSCs were isolated. And co-cultured at a ratio of 2∶1 and introduced into a silk fibroin (SF)/collagen scaffold to construct a tissue-engineered nerve conduit (TENC), which were transplanted to bridge 10 mm long sciatic nerve defects in rats. The experiment was divided randomly into 4 groups (10 rats/group): those bridged with plain SF/collagen scaffolds (Scaffold group), those bridged with TENCs (TENC group), those bridged with autografts (Autograft group) and those unoperated side (Normal group). The mechanical properties were examined using a universal testing machine(Instron 5865). Scanning electron microscopy was performed to observe the structure of the SF/collagen scaffold and the cells' growth. A series of electrophysiological examinations and morphological analyses were performed 12 weeks after surgery to evaluate the effect of the TENC on peripheral nerve regeneration.And One-way ANOVA was used to ana-lyze the data. If the differences between groups were statistically significant, the Turkey's method was further applied for comparison. Results The plain SF/collagen scaffold showed appropriate pore size and good intercommunicating of holes. The cells were tightly attached to and partly coiled about the scaffold and exhibited either a spindle or a spherical shape. The results of the mechanical measurement revealed that the maximum and average Young’s moduli of the SF/collagen scaffold were (10.80 ± 0.30) MPa and (8.14 ± 0.20) MPa, respectively. The mechanical properties ensure that the scaffold could resist muscular contraction and maintain its shape unchanged for a considerable period of time after grafting.All rats in each group had achieved nerve defect regeneration in varying degrees.But in terms of the effect of the repaired nerve, those treated with TENC were similar to those with autologous nerve grafts but superior to those with plain SF/collagen scaffolds. Conclusion The TENC that combined silk fibroin/collagen scaffold with a co-culture of SCs and ADSCs had normal nerve-like structure, and can bridge sciatic nerve defect and promote nerve growth.
RESUMO
Objective: To investigate the early effects of acellular xenogeneic nerve combined with adipose-derived stem cells (ADSCs) and platelet rich plasma (PRP) in repairing facial nerve injury in rabbits. Methods: The bilateral sciatic nerves of 15 3-month-old male Sprague-Dawley rats were harvested and decellularized as xenografts. The allogeneic ADSCs were extracted from the neck and back fat pad of healthy adult New Zealand rabbits with a method of digestion by collagenase type Ⅰ and the autologous PRP was prepared by two step centrifugation. The 3rd generation ADSCs with good growth were labelled with CM-Dil living cell stain, and the labelling and fluorescence attenuation of the cells were observed by fluorescence microscope. Another 32 New Zealand rabbits were randomly divided into 4 groups and established the left facial nerve defect in length of 1 cm ( n=8). The nerve defects of groups A, B, C, and D were repaired with CM-Dil-ADSCs composite xenogeneic nerve+autologous PRP, CM-Dil-ADSCs composite xenogeneic nerve, xenogeneic nerve, and autologous nerve, respectively. At 1 and 8 weeks after operation, the angle between the upper lip and the median line of the face (angle θ) was measured. At 4 and 8 weeks after operation, the nerve conduction velocity was recorded by electrophysiological examination. At 8 weeks after operation, the CM-Dil-ADSCs at the distal and proximal ends of regenerative nerve graft segment in groups A and B were observed by fluorescence microscopy; after toluidine blue staining, the number of myelinated nerve fibers in regenerated nerve was calculated; the structure of regenerated nerve fibers was observed by transmission electron microscope. Results: ADSCs labelled by CM-Dil showed that the labelling rate of cells was more than 90% under fluorescence microscope, and the labelled cells proliferated well, and the fluorescence attenuated slightly after passage. All the animals survived after operation, the incision healed well and no infection occurred. At 1 week after operation, all the animals in each group had different degrees of dysfunction. The angle θ of the left side in groups A, B, C, and D were (53.4±2.5), (54.0±2.6), (53.7±2.4), and (53.0±2.1)°, respectively; showing significant differences when compared with the healthy sides ( P0.05). At 8 weeks after operation, the fluorescence microscopy observation showed a large number of CM-Dil-ADSCs passing through the distal and proximal transplants in group A, and relatively few cells passing in group B. Toluidine blue staining showed that the density of myelinated nerve fibers in groups A and D were significantly higher than those in groups B and C ( P0.05). Transmission electron microscope observation showed that the myelinated nerve sheath in group D was large in diameter and thickness in wall. The morphology of myelin sheath in group A was irregular and smaller than that in group D, and there was no significant difference between groups B and C. Conclusion: ADSCs can survive as a seed cell in vivo, and can be differentiated into Schwann-like cells under PRP induction. It can achieve better results when combined with acellular xenogeneic nerve to repair peripheral nerve injury in rabbits.