Loading necrostatin-1 composite bone cement inhibits necroptosis of bone tissue in rabbit

Abstract

Bone necrosis after injecting of polymethylmethacrylate (PMMA) bone cement will lead to re-fracture of bone tissue. As a new type of necrosis, there is little research related to the necroptosis of surrounding bone tissue near the bone cement. The purpose of our study was to (i) investigate the presence of necroptosis in vivo and, (ii) established as a new type of bone cement containing PMMA, calcium phosphate cement (CPC) and Necrostatin-1 (Nec-1) to inhibit necroptosis of bone tissue. A total of 12 Japanese rabbits were used to establish the animal model and randomly divided into 4 groups signed as a control group, PMMA group, PMMA-CPC group and PMMA-CPC-Nec-1 group, respectively. We used scanning electron microscope to observe the structure of the samples, used HE staining to detect the necrosis, and used western blotting as well as ELISA test to examine the iconic molecule receptor interacting protein kinase-3 (RIP 3) protein and tumor necrosis factor α (TNF-α). After analyzing the results of our study, we found that the structure in both PMMA bone cement group and composite bone cement group was damaged and there was an evidence of necrosis, but it was absent in control group. Through molecule detection, the RIP 3 protein expression was decreased in PMMA-CPC-Nec-1 (P < 0.05). TNF-α expression was increased in bone cement groups with and without CPC (P < 0.05), but was inhibited in PMMA-CPC-Nec-1 group. We have concluded that the necroptosis could be confirmed in bone tissue necrosis induced by TNF-α after bone cement injection and also could be inhibited by composite bone cement with Nec-1.

Keywords: composite bone cement; necroptosis; necrostatin-1.

Figure 1

Process of establishing animal models: in Graph C, the red arrow points to the injection point, and black box is the sample intercept rang which centered on the injection point when it is used for protein detection (∼1 cm length and 0.5 cm width)

Figure 2

Flow cytometric analysis of MC3T3-E1 cell in vitro: (A) control group; (B) TNF-α group; (C) TNF-α plus sample releasing supernatant group; (D) analysis of necroptosis cells percentage.(* represents that there is significant difference between two groups, P < 0.05)

Figure 3

Electron microscopy scanning diagram of samples (magnified by 300 times): (A) control group; (B) PMMA; (C) PMMA+CPC; (D) CPC+PMMA+NEC-1

Figure 4

X-ray image and HE staining of the femurs(magnified by 20 times): (A) X-ray of the sample implanted bone cement (the red arrow indicates bone cement material injected into the bone tissue); (B) overall view of the HE staining sample (the blank part indicated by the red arrow is filled with PMMA material, the yellow arrow indicated the injection point, and the blue box is the observation area); (C) control group; (D) PMMA group: the black arrow shows the necrotic tissue; (E) PMMA+CPC group; (F) CPC+PMMA+NEC-1 group. In Graphs E and F, the black arrow shows the osteocytes were lost in the lacuna; (G) the comparison of percentage of necrotic cells between four groups (showed in C, D, E, F. * represents that there is significant difference between two groups, P < 0.05; NS shorts for there is no significant difference between two groups, P > 0.05)

Figure 5

The expression of RIP 3 protein by western blotting (* represents that there is significant difference between two groups, P < 0.05; NS shorts for there is no significant difference between two groups, P > 0.05)

Figure 6

The expression of RIP 3 protein by ELISA test (* represents that there is significant difference between two groups, P < 0.05; NS shorts for there is no significant difference between two groups, P > 0.05)

Figure 7

The ELISA test result of TNF-α protein expression (* represents that there is significant difference between two groups, P < 0.05; NS shorts for there is no significant difference between two groups, P > 0.05)