Elsevier

Biomedicine & Pharmacotherapy

Volume 99, March 2018, Pages 629-637
Biomedicine & Pharmacotherapy

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

https://doi.org/10.1016/j.biopha.2017.12.109Get rights and content

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.

Introduction

Bone is a vital regulated organ in locomotion, supporting organ and maintaining mineral homeostasis and structural robustness [1]. Due to the metabolic disorder, trauma, and cancer resection etc., bone defects such as atrophy and delayed bone regeneration often occurred [2]. Clinical treatments for compromised bone healing require multiple procedures and they are complex and costly [3]. It has been reported that over 5% of bone fractures fail to heal or demonstrate a delay in healing, leading to increased health cost and morbidity [4]. Therefore, effective strategies to improve bone healing are in great demand.

In recent years, scientists have paid more attention on mesenchymal stem cells (MSCs). Implantation of MSCs is considered to be an therapeutic strategy to improve bone healing as MSCs can proliferate and differentiate into multiple cells, including bone, cartilage, adipose, and muscle cells [4]. Bone marrow stromal cells (BMSCs), containing a small number of MSCs, is an increasingly important source of cells for cell therapy and tissue repair [5,6]. Through differentiating into selected cell types and secrete paracrine factor, BMSCs act as strong candidates for regulation of angiogenesis at the beginning of bone formation [7]. Previous study by Nishida et al. revealed that BMSCs protected and repaired damaged neurons through producing growth factors [8]. Another study by Wang et al. indicated that BMSCs induced bone formation when implanted with biodegradable scaffolding, suggesting a promising prospect for clinical treatments of bone damage [5].

Leucine-rich repeat-containing G-protein coupled receptor 6 (LGR6) belongs to leucine-rich repeat-containing subgroup of the G protein-coupled 7-transmembrane protein superfamily [9]. It is categorized into type B of LGR protein family, functioning in organogenesis and homeostasis in various tissues [10]. Along with the other two type B LGRs, i.e. LGR4 and LGR5, LGR6 is frequently expressed in distinct types of adult stem cells [11]. Specifically, LGR6 marked a group of stem cells in taste buds, lung and skin [12,13]. The study of LGR6 in stem cells thus becomes a hit. Evidences that confirm the significance of LGR6 have been accumulating. For instance, Lehoczky and Tabin found that type B LGRs marked in nail stem cells and could be a contributor to digit tip regeneration [14]. In addition, LGR6 promoted wound healing, angiogenesis, and nascent hair follicle development in damaged epithelial tissues [15]. However, not enough studies have been focused on the mechanism of LGR6 in BMSCs or its role in bone damage recovery.

In the present study, we used a microarray to explore the differentially expressed genes between fibroblasts and BMSCs. The role of LGR6 during the osteogenic differentiation were evaluated in vivo and vitro. After osteogenic induction, BMSCs with lower expression of LGR6 were found to highly express Collagen I, Runx2 (runt-related transcription factor 2), OCN (osteocalcin), ALP (alkaline phosphatas) and had a deeper mineralization, indicating that down-regulation of LGR6 promotes the osteogenic differentiation of BMSCs in vitro. Moreover, transplantation of LGR6-knockout BMSCs facilitated the healing of bone fracture in vivo, providing novel clinical therapy possibilities for bone healing.

Section snippets

BMSCs isolation and culture

Marrow samples used for BMSCs isolation were obtained from 11 patients undergoing hip surgeries (Female = 6, Male = 5, Age = 7–25) at the First Hospital of Lanzhou University. All patients were informed the consent and the present study was approved by the ethical committee of the First Hospital of Lanzhou University. BMSCs isolation was performed as previous reported [7,8]. In brief, bone marrow obtained from patients under sterile conditions was filtered with a cell strainer (Thermo Fisher

Microarray analysis and the differentially expressed genes

In present study, the mRNA expression profiling dataset was GSE92640, which was generated by Liu et al. using GPL6480 whole human genome microarray. To investigate the potential factors that induce the transformation of BMSCs into fibroblasts, the differentially expressed genes between BMSCs and fibroblast cells were analyzed using R software. A total of 162 differentially expressed genes were identified in the BMSCs group compared with the fibroblast cells group (Fig. 1A), the heat map showed

Discussion

The result of present study showed that LGR6 was overexpressed in BMSCs than in the fibroblasts. During the process of BMSCs osteogenic differentiation, expression of LGR6 gradually decreased. Furthermore, knocking-down of LGR6 promoted the osteogenic differentiation of BMSCs in vitro. Injecting the LGR6 knocked-down BMSCs into the defect site of rat fracture healing model has the potential to augment bone regeneration. Biomechanical tests demonstrated a statistically significant increase in

Author contributions

Research conception and design: Yingzhou Wang.

Data analysis and interpretation: Li Zhu and Renchun Huang.

Statistical analysis: Renchun Huang and Yanchao Cui.

Drafting of the manuscript: Yanchao Cui and Renchun Huang.

Critical revision of the manuscript: Li Zhu and Xueliang Zhang.

Approval of final manuscript: all authors.

Funding

This study was supported by the Science Foundation of China (No. H81371943).

Ethics approval and consent to participate

This study was approved by the ethical committee of the First Hospital of Lanzhou University and all participants signed the informed consent.

Consent to publish

Consent for publication was obtained from the participants.

Conflict of interest

None.

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