Abstract:
Background Microtissue is formed by the aggregation of seed cells under the action of cell-cell or cell-ECM (extracellular matrix). This 3D-cultured method is conducive to improve cell activity and tissue repair effect. However, microtissue is rarely used in neural tissue engineering.
Objective To construct adipose-derived mesenchymal stem cells (ASCs) into microtissues and explore the early repair effect of microtissues on rat sciatic nerve defect model.
Methods ASCs were extracted and identified. The microtissue was constructed by hanging-drop method or low-adhesion cell culture plate, and its properties were studied. The microtissue and the dorsal root ganglion (DRG) were co-cultured directly or indirectly, and the effect of microtissue on the growth of DRG axons was observed. Polycaprolactone (PCL) nerve conduit was constructed by electrospinning mechanism and its orientation was verified. Finally, 24 SD rats were randomly divided into hollow conduit group (hollow), 2D cells group (2D), microtissues group (microtissue) and autologous nerve graft group (ANG), with 6 rats in each group. The 1 cm defect model of sciatic nerve was constructed. The ASCs culture medium was injected after PCL conduit bridging, 2D ASCs were injected after PCL conduit bridging, microtissues were injected after PCL conduit bridging, and autologous nerve reverse transplantation were performed for four groups. The nerve grafts were removed for histological evaluation at 4 weeks after transplantation.
Results ASCs were successfully extracted and expressed mesenchymal stem cell surface marker proteins CD73, CD90 and CD105. After 7 days of dynamic observation, the diameter of microtissue was positively correlated with the number of cells, and with the extension of culture time, the microtissue showed a "compacted" state, and the diameter gradually decreased. In the indirect co-culture system of microtissue and DRG, the axon length of microtissue group was significantly longer than that of 2D group (P＜0.05) and control group (P＜0.05). In the direct co-culture system of microtissue and DRG, the axons emitted by DRG were closely connected with the nearby microtissues. PKH26 labeled ASCs were aligned on PCL film. The results of animal experiments showed that the axon growth of microtissue group was significantly better than that of 2D group (P＜0.05) and hollow group (P＜0.05).
Conclusion Microtissue can promote peripheral nerve regeneration and achieve good repair effect in the rat model of sciatic nerve defect.
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