Experimental Study Of Cannabinoid Receptor 2 In Titanium Particle Induced Osteolysis

Objective: To develop a titanium particle induced murine air-pouch model of bone resorption to evaluate the pathogenesis of aseptic loosening.Methods: Three millilitre sterilized air was injected subcutaneously to form an air pouch on the back of 20 female BALB/c mice, which were 6～8 weeks old and weighted 20～23g at the beginning of this study. The air pouches were injected with 1 ml air every other day for 5 days. At the 6th days, a section of calvaria bone from a genetically identical donor mouse, was implanted into the pouch. Then the mice were randomized into control groups (10 mice) and experimental groups (10 mice). In control groups, the saline (0.5ml) was injected into the pouch; in experimental groups, 5mg titanium particles suspended in 0.5ml saline were injected into pouches to provoke inflammation and osteolysis. At the 14th day after bone implantation, the mice were sacrificed and tissues were harvested. Then the histology and Tartrate-Resistant Acid Phosphatase ( TRAP ) Staining were carried out. RANK,RANKL and CPK gene levels were measured by Real-time quantitative reverse transcription polymerase chain reaction.Results: Bone implants were tolerated well in experiment mice, and no animal died within this study. Titanium particles-stimulated pouches exhibited pronounced erythematous and oedematous changes compared with control groups. The histological analysis showed that the pouch membrane were thicker and more cellular infiltration, compared with control groups. Intensive TRAP Staining was identified in experimental groups. Expression of osteolysis-related gene RANK, RANKL and CPK in air pouch tissue were at high levels when stimulated with titanium particles. Conclusion: This air-pouch bone resorption model induced by titanium particle has the advantage of sensitive, rapid, economical and reproducible. This model can mimic the pathogenesis of aseptic loosening, and provides a useful tool to investigate osteolysis induced by wear particle.Objective: To explore the mechanism of cannabionid receptor 2 in periprosthetic osteolysis and provide a new target for the prevention and treatment of aseptic loosening.Methods: Female BALB/c mice, 6-8 weeks old, were obtained from the laboratory animal research center of soochow university. Each of the mice weighted 20-23g at the beginning of this study. All the experiment animals were randomized into 3 groups, control group, titanium particles group and treatment group; and each experimental group was comprised of 10 mice. The titanium particle-induced murine air-pouch osteolysis model were established according to the process in partⅠ. Cannabinoid receptor 2 selective antagonist-AM630 (200μg/kg/d) was given to mice intraperitoneally 1th day after titanium particles introduction and maintained until the sacrifice of the mice. Pouch tissues were collected 14 days after titanium particles introduction for molecular and histological analyses. The pouch membrane thickness and cell infiltratio were tested by computerized image analysis system according to HE staining. Bone resorption was measured using Van Gieson staining. Osteoclast-like cells in the pouch memebrane were determined using Tartrate-Resistant Acid Phosphatase (TRAP) staining. Effects of AM630 on osteoclast apoptosis were detected by deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) analyses. Western blot was used to test the effects of AM630 on ERK and PI3K/Akt pathways. Protein expression of CB2, IL-1β, TNF-αand RANKL in the osteolysis tissue were tested by Immunohistological staining. CB2, IL-1β, TNF-α, CPK, RANKL and RANK gene mRNA levels were measured by Real-time quantitative reverse transcription polymerase chain reaction.Results: Titanium-particle-stimulated pouches exhibited apparent erythematous and oedematous changes compared with control pouches. AM630 treatment could mitigate this changes around bone implants. HE staining showed the pouch memebrane were more thicker and more macrophage cellular infilation, compared with the control and AM630 treatment groups. Histomorphometry analysis showed that the difference of pouch membrane thickness among three groups was in significant. The results of Van Gilson histological stains showed there were eclipsed changes at bone surfaces in contact with pouch membrane. In contrast, bone surface of control and treatment groups remained histological normal. Intensive TRAP Staining was identified in titanium particle-stimulated groups, and AM630 markedly reduced the number of TRAP-positive cells in pouch tissues. AM630 treatment could significantly increase the apoptosis cells in pouch membrane, compared with the other two groups. Stimulating with titanium particles markedly increased the phosphorylation of ERK and Akt. However, The increase in ERK and Akt phosphorylation was inhibited by the treatment with AM630. The results of Immunohistological staining demostrated that AM630 could reduce the higher expression of CB2, IL-1β, TNF-αand RANKL stimulated with titanium particle. Expression of CB2, IL-1β, TNF-α, CPK, RANKL and RANK gene in air pouch tissue were at a high level after titanium particle stimulation. However, the mRNA levels of these gene were markedly reduced when treated with AM630 for 14 days.Conclusions: This study confirmed that suppression the activation of cannabinoid receptor 2 with AM630 could inhibit the process of titanium particles-stimulated inflammatory osteolysis in a murine model. Therefore, cannabinoid receptor 2 selective anagonist represent a suitable therapeutic cannadidate for the prevention and treatment of aseptic loosening.