Study For The Inhibitory Effection Of Strontium On Titanium Particle-induced Inflammatory Osteolysis

Part one: Inhibitory effects of strontium on osteoclatogenesis induced bytitanium particlesObjective To observe the effect of strontium (Sr2+) on titanium (Ti) particles-inducedosteoclastgogenesis．Methods We selected murine macrophage cell line RAW264.7as the object of thestudy, using receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL;70ng/ml)and macrophage colony stimulating factor (M-CSF;30ng/ml)to induce RAW264.7toosteoclast differentiation. The experiment involved5groups：blank group, Ti group, Rgroup, R+Ti group and Sr2+group. The effect of Ti and (or) Sr2+on RAW264.7cellviability was measured by using the cell counting kit-8(CCK-8)．Tartrate resistant acidphosphatase(TRAP) staining was used to analyze the osteoclast formation in group．Thebone resorption activity of osteoclast was measured by osteo assay surface (OAS) plate.Reverse-transcription polymerase chain reaction (RT-PCR) was used to detect the mRNA1evels of cathepsin K (Cath-K)， TRAP, matrix metalloproteinase-9(MMP-9), calcitoninreceptor (CTR)．Results Sr2+(0.5、1、2、5、10mmol/L) did not affect the viability of RAW264.7cellscultured with Ti particles. The mature osteoclasts were obtained from RAW264.7cellscultured with RANKL and Ti particles for5days and detected by TRAP staining. Thenumber of TRAP positive cells in5mmol/L Sr2+was (323.33±43.84)/well which wassignificantly decreased as compared with R group[(453.33±33.99)/well](P<0.05)．OASrevealed that the area of bone resorption pits in5mmol/L Sr2+group was（6.53±2.02）%,incombination with R+Ti group [（47.24±5.03）%](P<0.05). RT-PCR demonstrated thatmRNA levels of Cath-K, CTR, MMP-9and TRAP were significantly greater in mediumwith RANKL and Ti particles than without RANKL and Ti particles. When5mmol/L Sr2+was added to the culture system for5days, the mRNA levels of Cath-K、TRAP、MMP-9 and CTR respectively were2.76±0.66,2.69±0.57,2.10±0.34and1.72±0.32, incombination with RANKL and Ti particles (7.97±0.77,10.10±1.18,7.37±1.02and5.55±0.53)(P<0.05)..Conclusion This study showed that Sr2+can significantly inhibit Ti particle-inducedthe expression of Cath-K, TRAP, MMP-9and CTR, inhibit osteoclastogenesis andalleviate the osteoclatic resorption in vitro.Part two: Study for the mechanism of the inhibitory effects of strontiumon titanium particles-induced osteoclatogenesisObjective To explore the effect of Sr2+on NF-κB signaling pathway and theexpression of inflammatory cytokines in the process of Sr2+inhibiting titanium particlesinduced-osteoclastogenesis.Methods We selected murine macrophage cell line RAW264.7as the object of thestudy. The experiment is divided into three groups: blank group, Ti group andTi+Sr2+group. GroupC: Ti+Sr2+group. The inhibitory κBα (IκBα) in NF-κB signalingpathway was determined by Western blot. The mRNA1evels of nuclear factor of activatedT-cells c1(NFATc1) and c-fos was detected by reverse-transcription polymerase chainreaction．The amounts of interleukin-1β (IL-1β), interleukin-6(IL-6) and tumor necrosisfactor-α (TNF-α) produced in the supernatants were analyzed using mouse-specificenzyme linked immunosorbant assay (ELISA) kits.Results Western blot showed that the levels of IκBα was reduced at15min butincreased again at180min after Ti particle treatment. Pretreatment with Sr2+significantlyincreased the expression of IκBα in response to Ti particle. RT-PCR revealed that Tisignificantly up-regulated the mRNA expression levels of NF-κB two target transcriptionfactors, NFATc1and c-fos. Pretreatment with Sr2+for120min and then cultured with Sr2+for6h, the mRNA levels of NFATc1and c-fos at60min respectively were2.44±0.46and2.53±0.22，in combination with Ti particles group(4.72±0.46and4.34±0.53)(P<0.05). Inaddition, ELISA detected that the protein secretion levels of TNF-α, IL-1β and IL-6in Tigroup at48h respectively were (309.6±20.0)pg/ml，(257.6±14.3)pg/ml and (164.4±13.1)pg/ml，in combination with Ti+Sr2+group [(153.0±14.3),(203.2±13.5)and (101.4±9.1)] pg/ml (P<0.05). Conclusion This study showed that Sr2+can significantly inhibit Ti particle-inducedosteoclastogenesis by disturbing the NF-κB signaling pathway and the expression ofinflammatory cytokines.Part three: Study for the inhibitory effection of strontium ranelate ontitanium particles-induced inflammatory osteolysisObjective To observe the effect of strontium ranelate (SR) on titanium particles(Ti)-induced inflammatory osteolysis in vivo.Methods In our study, we used sixty male C57BL/J6mice，8-10weeks old, toobserve the degree of bone resorption in a murine osteolysis model. All experimentalanimals were randomly divided into four groups：blank group, SR group, Ti group, Ti+SRgroup. Once Ti particles were implanted in mice, the mice were administrated SR(900mg/kg/d) by gavage for14days. After14days, the calvaria were collected formicro-computed tomography (Micro-CT) analysis, histological staining and proteindetection.Results The results of Micro-CT analysis showed the bone minaral density (BMD),bone volume (BV) and bone volume/tissue volume (BV/TV) of the calvaria respectivelywere (0.73±0.03) mg/mm2,(1.50±0.03) mm3and (21.97±0.55)%, in combination withTi group [(0.62±0.03) mg/mm2,(1.22±0.07) mm3,(16.05±0.63)%](P<0.05). Theanalysis of HE staing demonstrated that SR markly reduced the thickness of the periosteumand the area of bone resportion in the presecece of Ti in vivo. TRAP staing indicated thatSR significantly inhibited the generation of tartrate-resistant acid-phosphatase（TRAP）positive cells in a murine osteolysis model, when compared with Ti stimulated calvaria.Immumohistochemical staining（IHS） showed that SR inhibited the expression of thereceptor activators of nuclear factor-kappa B (RANK) and tumor necrosis factor-α(TNF-α) in Ti particle-charged calvaria. In addition，enzyme linked immunosorbant assays(ELISA) analysis detected that the protein levels of receptor activators of nuclear factor-kappa B ligand (RANKL), osteoprotegerin (OPG), TNF-α and interleukin-1β (IL-1β) incalvaria of Ti+SR group group respectively were （2050±42）pg/ml,（13.0±1.0）pg/ml,（145.6±14.2）pg/ml and（130.2±8.2）pg/ml，in combination with Ti group [(1636±61),（19.6±1.8）,（210.2±8.9）and（159.6±9.7）] pg/ml (P<0.05). Conclusion This study showed that SR can obviously inhibit Ti particle-inducedinflammatory osteolysis by restraining osteoclastogenesis via regulatingRANKL/RANK/OPG and reducing the secretion of TNF-α and IL-1β.