Dorsal Root Ganglion Maintains Stemness of Bone Marrow Mesenchymal Stem Cells by Enhancing Autophagy through the AMPK/mTOR Pathway in a Coculture System.

Shuaishuai Zhang,Yi Gao,Pengzhen Cheng,Hao Wu,Yue Song,Chunmei Wang,Bin Liu,Tianqing Cao,Huijie Jiang,Junqin Li,Guoxian Pei,Jimeng Wang,Donglin Li,Liu Yang

doi:10.1155/2018/8478953

Abstract

Our previous studies found that sensory nerve tracts implanted in tissue-engineered bone (TEB) could result in better osteogenesis. To explore the mechanism of the sensory nerve promoting osteogenesis in TEB in vitro, a transwell coculture experiment was designed between dorsal root ganglion (DRG) cells and bone marrow mesenchymal stem cells (BMSCs). BMSC proliferation was determined by CCK8 assay, and osteo-, chondro-, and adipogenic differentiation were assessed by alizarin red, alcian blue, and oil red staining. We found that the proliferation and multipotent differentiation of BMSCs were all enhanced in the coculture group compared to the BMSCs group. Crystal violet staining showed that the clone-forming ability of BMSCs in the coculture group was also enhanced and mRNA levels of Sox2, Nanog, and Oct4 were significantly upregulated in the coculture group. Moreover, the autophagy level of BMSCs, regulating their stemness, was promoted in the coculture group, mediated by the AMPK/mTOR pathway. In addition, AMPK inhibitor compound C could significantly downregulate the protein expression of LC3 and the mRNA level of stemness genes in the coculture group. Finally, we found that the NK1 receptor antagonist, aprepitant, could partly block this effect, which indicated that substance P played an important role in the effect. Together, we conclude that DRG could maintain the stemness of BMSCs by enhancing autophagy through the AMPK/mTOR pathway in a transwell coculture system, which may help explain the better osteogenesis after implantation of the sensory nerve into TEB.

Highlights

Bone tissue engineering has provided a promising resolution for the treatment of large bone defect, there are still many problems to be solved [1,2,3], such as low survival rate and poor osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs)
Our results indicated that dorsal root ganglion (DRG) could help maintain the stemness of BMSCs through improving the basal autophagy level by activating AMPK/mTOR signaling in this coculture system
The multipotential differentiation of BMSCs was verified by alizarin red staining (Figure 1(b)), oil red O staining (Figure 1(c)), and alcian blue staining (Figure 1(d)), which showed that BMSCs used in this study could differentiate into osteoblasts, adipocytes, and chondrocytes under induction condition

Summary

Introduction

Bone tissue engineering has provided a promising resolution for the treatment of large bone defect, there are still many problems to be solved [1,2,3], such as low survival rate and poor osteogenic differentiation of BMSCs. The sensory nerve has been reported to play critical roles in bone metabolism and regeneration in vivo [6,7,8,9]. Some studies found that sensory nerve innervation contributed to the maintenance of trabecular bone mass and its mechanical properties by inhibiting bone resorption [8]. Sensory nerves had efferent functions in the tissues they innervated, mediated by transmitters released from the peripheral nerve terminals, which helped to maintain trabecular bone integrity [10]. Sensory neuron-derived Sema3A was found to be responsible for the bone mass loss

Methods

Results

Conclusion