Experimental Study Of The Inhibitory Effect Of Titanium Particles On Heterotopic Ossification

ObjectiveTo induce heterotopic ossification through traumatic animal model and to implant the titanium particles into the area where would form the heterotopic bone.To investigate the possible mechanism for this treatment method.MethodsThirty-six kunming strain mice were used to establish traumatic animal models by Achilles tenotomy and injecting 0.2 mL suspension of titanium particles into the left Achilles and the normal sodium to the other limb.X-ray, histological examination and immunohistochemistry were made at 4,8,12 weeks respectively.Results1. Under X-ray examination:by 8 weeks,new bone(radio-opaque zones)in the region of the Achilles tendon was found in 58.3% and in 100% by 12 weeks.2.This heterotopic ossification occured through a process of endochondral ossification. 3.Immunohistoehemistry result:①The expression of BMP-2,TGF-βshow stronger in the control group than that in experimental group(P>0.05);②The expression of IL-1,TNF-a decreased with the time,and the experimental group was much higher than that in the contral group(P<0.05);③The expression of Runx2 in the experimental group was lower than that in the contral group;.④The expression of MMP-9 in the experimental group is significantly higher than that in contral group especially after 12 weeks(P<0.05).Conclusion1. Achilles tenotomy is a simple and feasible method to induce heterotopic ossification.2. The local use of titanium particles is a effective way to inhibit the formation of endochondral ossification. Objective1.To clarify the role of titanium particles on inflammatory cytokines releasing in macrophage cells.2.To explore the mechanism of titanium particles on the formation of osteoclast from osteoclast precursors (RAW264.7), and provide more evidences for titanium particles on the treatment of formation of hetero-topic ossification.Methods1.Macrophages were cocultured in vitro with cleaned titanium particles, TNF-α,IL-1, IL-6 and PGE2 in the supernatants were assayed after culturing for 24,48 and 72 hours.2. RT-PCR and Western blotting were used to examine the mRNA and protein expression of NFATc1 in titanium particle-induced RAW264.7 undergoing differentiation into osteoclasts.Results1. In the experimental group,TNF-α,IL-1,IL-6和PGE2 content was gradually increased, compared with the control group, there is a significant difference (P<0.05)2. The protein of NFATc1 as well as mRNA expressed by RAW264.7 was enhanced by stimulation of titanium particles.Conclusion1.The macrophages are sensitive to titanium particles to release inflammatory cytokines.2.RAW264.7 was differentiate to osteoclast by stimulation of titani-um particles. ObjectiveTo evaluate the effect of titanium particles on the proliferation, differentiation, mineralization and Runx2/Cbfα1, OPG, RANKL in MC3T3-E1,then investigate the possible mechanism of titanium particles on bone metabolism.Methods1. MC3T3-E1 cells were cultured for 22 days,MTT test was used to assess the cell proliferation at 2,4,7,14 days; while alkaline phospha-tase(ALP) staining and ALP activity measurement were taken to assess the differentiation of osteoblasts at 7,14 days. The mineralization of osteoblasts was evaluated by Alizarin red staining at 14,21 days.2. The expressions of Runx2/Cbfα1 mRNA and protein were tested using RT-PCR and Western blot respectively.3. MC3T3-E1 cells were cultured with different dose of titanium particles,the expressions of OPG and RANKL were obtained with RT-PCR and Western blot method.Results:1.The osteoblastic proliferation had significant difference between the experimental group and the control group (P<0.05),the proliferation of experimental group is inhibited by titanium particles. However, the colleganⅠstaining was weaken in the experimental group than that of control group at 14 days, meanwhile,activity of ALP measurement in the experimental group was smaller than the control group (P<0.01), while the alizarin red staining demonstrated that the quantity of calcium nodules were markedly decreased in the experimental group at 21 days.2. Titanium particles down-regulated the expression of Runx2/Cbfα1 at both mRNA and protein level at anytime.3.The titanium particles induced a constant and varying increases in the OPG and RANKL, and the upregulated mRNA and protein level of RANKL was higher than that of OPG.The RANKL/OPG ratio is in accordance with dose increase of the titanium particlesConclusion:1.The proliferation,differentiation and mineralization of primary rat osteoblasts are decreased with exposure to titanium particles2.The negative effect of titanium particles on differentiation of MC3T3-E1 cells may is related to its down-regulated the expression of Runx2/Cbfα13.The effects of titanium particles on osteoclast may is related to its regulating effect on the OPG/RANKL pathway of osteoblast.