Titanium particle‑mediated osteoclastogenesis may be attenuated via bidirectional ephrin‑B2/eph‑B4 signaling in�vitro

Abstract

The present study investigated the role of bidirectional ephrin-B2/erythropoietin-producing human hepatocellular receptor 4 (ephB4) signaling in the regulation of wear particle-mediated osteoclastogenesis in vitro. Mouse bone marrow macrophages (BMMs) were induced into osteoclasts by receptor activator of nuclear factor-κB ligand (RANKL, 50 ng/ml). EphB4-Fc, an osteoblast membrane surface receptor (4 µg/ml), was used to stimulate the ephrin-B2 ligand of osteoclasts in the presence and absence of titanium (Ti). Tartrate-resistant acid phosphatase (TRAP) staining was used to detect the number of osteoclasts, and phalloidin staining was used to examine the cytoskeletons of the osteoclasts. A bone pit absorption experiment was used to measure osteoclast function. Reverse transcription quantitative polymerase chain reaction and western blot analysis were used to examine osteoclastogenesis. ELISAs were used to detect the production of inflammatory factors. The data demonstrated that Ti significantly promoted the differentiation of BMMs into mature osteoclasts in the presence of RANKL and significantly promoted expression of the ephrin-B2, nuclear factor of activated T-cells 1 (NFATc1), TRAP, Fos proto-oncogene, AP-1 transcription factor subunit (C-FOS), and matrix metalloproteinase 9 (MMP9) genes. Phalloidin and TRAP staining revealed that following the addition of ephB4-Fc, the number, size and cytoskeletal elements of Key words: osteoclasts, osteoblasts, remodeling, ephrin-B2, osteoclastogenesis osteoclasts were significantly decreased compared with those in the titanium particle group without ephB4-Fc. Compared with the titanium particle group, the bone pit absorption experiment revealed significantly decreased absorption pit areas in the titanium particle+ephB4-Fc group. The expression of the NFATc1, TRAP, C-FOS and MMP9 genes was markedly decreased in the ephB4-Fc group; however, the expression of the ephrin-B2 gene was increased compared with the Ti particle group without ephB4-Fc after 5 days. Production of inflammatory cytokines was inhibited by Ti particles through bidirectional signals. Addition of ephB4-Fc inhibited the osteoclast-mediated formation of Ti particles via bidirectional ephrin-B2/ephB4 signaling. Activation of this bidirectional signaling pathway may be a potential clinical treatment for osteolysis surrounding prostheses.

The Recombinant Protein EphB4-Fc Changes the Ti Particle-Mediated Imbalance of OPG/RANKL via EphrinB2/EphB4 Signaling Pathway and Inhibits the Release of Proinflammatory Factors In Vivo

“…A chromogenic end-point TAL with Diazo coupling kit (Xiamen Houshiji, Fujian, China) was used to detect endotoxins [27]. The wear particles can be used for experiments until the endotoxin unit (EU) concentration is <0.1 EU/mL [27]. This concentration was similar to that of the wear particles around the prosthesis, as reported previously [1,7].…”

“…Ti particles were prepared as described previously [1]. We followed the methods of Dr. Ge et al 2018 [27]. The wear particles were soaked in 75% (v/v) ethanol for 48 hours to remove endotoxins [27].…”

“…We followed the methods of Dr. Ge et al 2018 [27]. The wear particles were soaked in 75% (v/v) ethanol for 48 hours to remove endotoxins [27]. A chromogenic end-point TAL with Diazo coupling kit (Xiamen Houshiji, Fujian, China) was used to detect endotoxins [27].…”

“…The wear particles were soaked in 75% (v/v) ethanol for 48 hours to remove endotoxins [27]. A chromogenic end-point TAL with Diazo coupling kit (Xiamen Houshiji, Fujian, China) was used to detect endotoxins [27]. The wear particles can be used for experiments until the endotoxin unit (EU) concentration is <0.1 EU/mL [27].…”

“…All animal procedures were approved by the Animal's Hospital affiliated to Shanghai Jiao Tong University. Bone marrow macrophages (BMMs) were collected from the tibias and femurs of 4-6-week-old C57BL/6 mice, containing 30 ng/mL M-CSF, 10% FBS, and 1% penicillinstreptomycin at 37°C for 3 days [27]. At~80% confluence, the cells were dissociated with 0.25% (v/v) trypsin and then plated onto culture plates.…”