No abstract

A previous study showed that BMP-2 (bone morphogenetic protein-2) and wear debris can separately support osteoclast formation induced by the receptor activator of NF-κB ligand (RANKL). However, the effect of BMP-2 on wear debris-induced osteoclast formation is unclear. In this study, we show that neither titanium particles nor BMP-2 can induce osteoclast formation in RAW 264.7 mouse leukemic monocyte macrophage cells but that BMP-2 synergizes with titanium particles to enhance osteoclast formation in the presence of RANKL, and that at a low concentration, BMP-2 has an optimal effect to stimulate the size and number of multinuclear osteoclasts, expression of osteoclast genes, and resorption area. Our data also clarify that the effects caused by the increase in BMP-2 on phosphorylated SMAD levels such as c-Fos expression increased throughout the early stages of osteoclastogenesis. BMP-2 and titanium particles stimulate the expression of p-JNK, p-P38, p-IkB, and P50 compared with the titanium group. These data suggested that BMP-2 may be a crucial factor in titanium particle-mediated osteoclast formation.

To investigate the association between primary knee osteoarthritis (OA) and single nucleotide polymorphism (SNP) (A668G) of leptin receptor gene (LEPR) in the Ningxia Hui population. A case-control association study has been adopted in this thesis. The polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis were performed to investigate the SNP of A668G site within LEPR from 148 patients with knee OA and 155 controls (asymptomatic and radiographically negative) with matched age and gender among Ningxia Hui population. In addition, genotypes of LEPR were verified by direct sequence analysis on PCR products. The result indicates that allele and genotype frequencies (P=0.024 and 0.008, respectively) in LEPR SNP A668G were significantly different in the knee OA patients group and control group, and in the knee OA patients group, the serum levels of leptin decreased significantly (P<0.001) and the serum levels of soluble leptin receptor increased significantly (P<0.001) compared with control group. Therefore, LEPR SNP A668G is associated with susceptibility to knee OA, which would be used as the genetic marker in predicting the risk of knee OA and would be one of the candidate genes in early prevention and control.

The phagocytosis of wear particles by macrophages results in the secretion of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), which play a major role in promoting osteoclast recruitment. The inhibition of TNF-α expression decreases osteoclastogenesis. In a previous study, we demonstrated that bone morphogenetic protein-2 (BMP-2) can activate wear debris-induced osteoclast recruitment in the presence of receptor activator of nuclear factor (NF)-κB ligand (RANKL); however, whether these effects are associated with pro-inflammatory cytokines remains unclear. In this study, we constructed an adenoviral vector carrying TNF-small interfering RNA (siRNA) (Ad-TNF-siRNA), as well as a vector carrying both the BMP-2 gene and TNF-α-siRNA (Ad-BMP-2-TNF-siRNA). The two adenoviral vectors significantly suppressed the expression of TNF-α; however, only treatment with Ad-TNF-siRNA significantly inhibited osteoclastogenesis. We demonstrate that the overexpression of BMP-2, despite the suppression of TNF-α expression by Ad-BMP-2-TNF-siRNA, increases the size and number of titanium (Ti) particle-induced multinuclear osteoclasts, the expression of osteoclast genes, as well as the resorption area. There were no differences observed between Ti particle-induced and Ad-BMP-2-TNF-siRNA-induced osteoclast formation. Moreover, Ad-BMP-2-TNF-siRNA directly acted upon osteoclast precursors by increasing the level of c-Fos, regulating other signaling pathways, such as p38 phosphorylated c-Jun N-terminal kinase (p-JNK) and phosphorylated IκB (p‑IκB). Taken together, these data demonstrate that treatment with Ad-BMP-2-TNF-siRNA increases wear debris-induced osteoclast formation by activating c-Fos and that these effects are not associated with pro-inflammatory cytokines.

Wear particles are phagocytosed by macrophages and other inflammatory cells, resulting in cellular activation and release of proinflammatory factors, which cause periprosthetic osteolysis and subsequent aseptic loosening, the most common causes of total joint arthroplasty failure. During this pathological process, tumor necrosis factor-alpha (TNF-α) plays an important role in wear-particle-induced osteolysis. In this study, recombination adenovirus (Ad) vectors carrying both target genes [TNF-α small interfering RNA (TNF-α-siRNA) and bone morphogenetic protein 2 (BMP-2)] were synthesized and transfected into RAW264.7 macrophages and pro-osteoblastic MC3T3-E1 cells, respectively. The target gene BMP-2, expressed on pro-osteoblastic MC3T3-E1 cells and silenced by the TNF-α gene on cells, was treated with titanium (Ti) particles that were assessed by real-time PCR and Western blot. We showed that recombinant adenovirus (Ad-siTNFα-BMP-2) can induce osteoblast differentiation when treated with conditioned medium (CM) containing RAW264.7 macrophages challenged with a combination of Ti particles and Ad-siTNFα-BMP-2 (Ti-ad CM) assessed by alkaline phosphatase activity. The receptor activator of nuclear factor-κB ligand was downregulated in pro-osteoblastic MC3T3-E1 cells treated with Ti-ad CM in comparison with conditioned medium of RAW264.7 macrophages challenged with Ti particles (Ti CM). We suggest that Ad-siTNFα-BMP-2 induced osteoblast differentiation and inhibited osteoclastogenesis on a cell model of a Ti particle-induced inflammatory response, which may provide a novel approach for the treatment of periprosthetic osteolysis.