LCPD is an idiopathic osteonecrosis, which is mainly manifested by necrosis of femoral head and cartilage induced by interruption of blood supply, resulting in gradual deformity and degenerative osteoarthritis [1]. It is one of the common puzzle diseases in orthopedics, mainly seen in children aged 5 to 7 [2]. The annual incidence rate of LCPD is about 5 cases per 1,000,000- to 14-year-old children [3]. Healing usually leaves various degree of deformity and joint dysfunction, which may develop into early osteoarthritis. The etiology of LCPD is not very clear at present. It is generally believed that its pathogenesis is related to blood supply of femoral head, lesions around hip joint, trauma, endocrine factors and so on [4]. Therefore, how to effectively promote the repair of femoral head necrosis, avoid the excessive absorption of necrotic bone, promote the formation of new bone and improve the prognosis has become a challenge and problem.

Previous studies have confirmed that LCPD displayed chronic inflammation, especially characterized by early-onset chronic synovitis of the hip. The researchers also found that the expression levels of inflammatory cytokines in necrotic bone tissue increased significantly, which activated the corresponding inflammatory signals and further aggravated the disease [5]. Macrophages are essential for host defense and inflammatory response. According to the perceived microenvironment stimuli, they differentiate into two different polarization phenotypes and show different functions: classically activated M1 macrophages and alternatively activated M2 macrophages. M1 has a pro-inflammatory effect via secreting pro-inflammatory factors, while M2 macrophages highly express CD206, IL-10, IL-13, IL-4, IL-5 and other anti-inflammatory cytokines or genes such as Aginase 1 (Arg-1) to prevent excessive inflammation and promote tissue repair [6], [7], [8]. Toll like receptor 4 (TLR4) is one of the critical members of pattern recognition receptors expressed by macrophages. Necrotic bone tissue can stimulate M1 macrophages to secrete pro-inflammatory cytokines and inhibit new bone formation through TLR4 signal pathway [9]. Meanwhile, paclitaxel has been shown to reprogram M2-polarized macrophages into an M1-like phenotype via TLR4 activation [10]. In addition, inhibition of TLR4 is found to promote M2 macrophages polarization and accelerate wound healing process [11].

It has been reported that miR-214-3p was downregulated in femoral head cartilage tissue, serum and chondrocytes from LCDP samples, and miR-214-3p overexpression enhanced cell activity of human chondrocyte cell line TC28 by inhibiting Bax [12]. Therefore, miR-214-3p may act as a protective factor in LCPD. However, the effects and mechanisms of miR-214-3p in LCPD remain unknown. miR-214-3p has been reported to promote M2 polarization of macrophages by targeting GSK3β in allergic rhinitis, suggesting that miR-214-3p may reduce inflammatory response and repair tissue damage [13]. Activation transcription factor 4 (ATF4), sponged by miR-214, plays a key role in bone formation, which regulates the proliferation and differentiation of chondrocytes during endochondral ossification. Nevertheless, whether miR-214-3p could take its actions on the regulation of macrophages polarization in LCPD pathogenesis through TLR4, remains unclear. In addition, on the basis that miR-214-3p had no direct targeting relationship with TLR4, activation transcription factor 7 (ATF7), previously regarded as a crucial role in controlling memory of macrophages, shared a potential complementary binding site with miR-214-3p, we proposed that miR-214-3p might modulate TLR4 at transcriptional level via affecting ATF7.

As an extracellular vesicle secreted by cells, exosomes carry proteins and RNAs for communication between cells. Studies also confirmed that exosomes secreted by chondrocytes promoted the maturation of IL-1β in macrophages and aggravated synovitis in osteoarthritis [14]. In addition, angiogenesis is an important process of new bone formation. Studies have also revealed that miR-214-3p in cartilage promoted endothelial cell migration and angiogenesis by regulating the paracrine VEGF of TrkB/ShcB pathway [15]. We also found a potential complementary binding site between miR-214-3p and RUNX1, which is identified to be a transcriptional inhibitor of VEGFA.

In this study, we isolated exosomes from chondrocytes and administrated with miR-214-3p to investigate the effects of macrophage polarization and angiogenesis in LCPD and the underlying mechanisms. Herein, we reported that miR-214-3p was poorly expressed in LCPD and overexpression of miR-214-3p could promote cell proliferation and inhibit apoptosis in DEX-treated TC28 cells. Furthermore, exos-miR-214-3p facilitated M2 polarization by targeting ATF7/TLR4 axis and HUVECs angiogenesis via regulating RUNX1/VEGFA axis. These results might provide new therapeutic targets and strategies for LCPD treatment.