Aims. MicroRNA-183 (miR-183) is known to play important roles in osteoarthritis (OA) pain. The aims of this study were to explore the specific functions of miR-183 in OA pain and to investigate the underlying mechanisms. Methods. Clinical samples were collected from patients with OA, and a mouse model of OA pain was constructed by surgically induced destabilization of the medial meniscus (DMM). Reverse transcription quantitative polymerase chain reaction was employed to measure the expression of miR-183, transforming growth factor α (TGFα), C-C motif chemokine ligand 2 (CCL2), proinflammatory cytokines (interleukin (IL)-6, IL-1β, and tumour necrosis factor-α (TNF-α)), and pain-related factors (transient receptor potential vanilloid subtype-1 (TRPV1), voltage-gated sodium 1.3, 1.7, and 1.8 (Nav1.3, Nav1.7, and Nav1.8)). Expression of miR-183 in the dorsal root ganglia (DRG) of mice was evaluated by in situ hybridization. TGFα, CCL2, and C-C chemokine receptor type 2 (CCR2) levels were examined by immunoblot analysis and interaction between miR-183 and TGFα, determined by luciferase reporter assay. The extent of pain in mice was measured using a behavioural assay, and OA severity assessed by Safranin O and Fast Green staining. Immunofluorescent staining was conducted to examine the infiltration of macrophages in mouse DRG. Results. miR-183 was downregulated in tissue samples from patients and mice with OA. In DMM mice, overexpression of miR-183 inhibited the expression of proinflammatory cytokines (IL-6, IL-1β, TNF-α) and pain-related factors (TRPV1, Nav1.3, Nav1.7, Nav1.8) in DRG. OA pain was relieved by miR-183-mediated inhibition of macrophage infiltration, and dual luciferase reporter assay demonstrated that miR-183 directly targeted TGFα. Conclusion. Our data demonstrate that miR-183 can ameliorate OA pain by inhibiting the TGFα-CCL2/CCR2 signalling axis, providing an excellent therapeutic target for OA treatment. Cite this article: Bone Joint Res 2021;10(8):548–557

Summary Statement. MCP-1/ CCR2 axis at the early phase plays a pivotal role in the fracture healing. Inflammation plays a pivotal role in fracture healing. Among them, chemokines play key roles in inflammation. Monocyte chemotactic protein-1 (MCP-1), via its receptor C-C chemokine receptor 2 (CCR2), acts as a potent chemoattractant for various cells to promote migration from circulation to inflammation site. Thus, the importance of MCP-1/CCR2 axis in fracture healing has been suggested. However, the involvement of MCP-1/CCR2 axis tofracture site is not fully elucidated. Results. PCR Array: The expression of MCP-1 and MCP-3 had increased on day 2 than 0 or 7 in the rib fracture healing. Immunohistochemistry Staining: To verify the localization of MCP-1 expression, we examined the Wild type (WT)-mouse rib fracture healing. We observed high expression of MCP-1 and MCP-3 at the periosteum and the endosteum on post-fracture day 3. In vivo Antagonist Study: To elucidate whether MCP-1/CCR2 axis is involved during the early phase of fracture healing, we continuously administered RS102895, CCR2 antagonist, before or after rib fracture. Micro-CT analysis showed delayed fracture healing in the before-group compared with both the control and after-group. On day 21, the hard callus volume in the before-group was significantly smaller than that in the control-group. Histological analysis showed that fractures in both the control and the after-groups were healed by day 21. In contrast, less of cartilage in the callus was observed in the before-group on day 7. Gain of Function: To examine the roles of MCP-1 at the periosteum and the endosteum during the fracture healing, we created a segmental bone graft exchanging model. The bone grafts were transplanted from MCP-1. −/−. mice to another MCP-1. −/−. mice (KO-to-KO). Micro-CT analysis showed that KO-to-KO transplantation led to the delay of fracture healing on day 21. Next, we created exchanging-bone graft models between MCP-1. −/−. and WT mice, in which a segmental bone derived from a WT mouse was transplanted into a host MCP-1. −/−. mouse (WT-to-KO). In contrast to KO-to-KO bone graft transplantation, the transplantation of WT-derived graft into host KO mouse resulted in a significant increase of new bone formation on day 21. Histological analysis revealed that marked and localised induction of MCP-1 expression in the periosteum and the endosteum around the WT-derived graft was observed in the host MCP-1. −/−. mouse. Loss of Function: To validate whether MCP-1 is a crucial chemokine for fracture healing, we created WT-to-WT and KO-to-WT bone graft models. When WT-donor graft was transplanted into WT-host, abundant new bone formation was observed around a WT-derived graft on day 21. In contrast, transplantation of KO-derived graft into WT-host resulted in a marked reduction of periosteal bone formation on a donor graft. Discussion. In this study, we demonstrated that MCP-1/ CCR2 axis at the early phase modulates the fracture healing. Furthermore, we showed that MCP-1 in the periosteum and the endosteum promotes the fracture healing in vivo. Thus, these results clearly suggest that MCP-1 in the periosteum and the endosteum at the early inflammatory phase is an essential component for successful fracture healing

Objectives

The objective of this study was to investigate the therapeutic effect of peripheral blood mononuclear cells (PBMNCs) treated with quality and quantity control culture (QQ-culture) to expand and fortify angiogenic cells on the acceleration of fracture healing.