Down-regulation of LGR6 promotes bone fracture recovery using bone marrow stromal cells

Abstract

Objective: The Leucine-rich repeat-containing G-protein coupled receptor 6 (LGR6) is a well-known marker of stem cells. In present study, we aimed to further explore the effects of LGR6 on promoting osteogenic differentiation of bone marrow stromal cells (BMSCs) and bone healing.

Methods: Flow cytometry assay was used to determine the expression of BMSCs surface markers, and western blot was performed to detect the LGR6 protein expression. The osteogenic differentiation of BMSCs was qualified using ALP and ARS staining. Protein expression of osteogenic genes (ALP, Collagen I, Runx2 and OCN) were evaluated using western blot. In vivo, BMSCs transfected with sh-LGR6 or LGR6 cDNA were injected into the fracture site to establish rat fracture healing model. X-ray system and hematoxylin-eosin (HE) staining were conducted to observe the fracture recovery. Biomechanical test was performed to detect the changes of maximum load, elastic modules and bone mineral density.

Results: In BMSCS, CD90 and CD44 were positively expressed, while CD11b was negatively expressed. Expression level of LGR6 gradually decreased with the osteogenic differentiation of BMSCs. The osteogenic genes expression level during the osteogenic differentiation significantly increased with the down-regulation of LGR6. In vivo, 8 weeks after injection, rats treated with LGR6 knocked-down BMSCs showed increased number of fibroblasts. Maximum load, elastic modulus and the bone mineral density were enhanced with the knocking-down of LGR6.

Conclusion: Inhibition of LGR6 promoted the osteogenic differentiation of BMSCs in vitro. Moreover, transplantation of LGR6-knockout BMSCs in rat models contributes to a better recovery after the fracture.

Keywords: BMSCs; Bone healing; LGR6.