Experiment Studies On The Effects Of Titanium Alloy Particle On Human Osteoblast In Vitro

Objective: To study the effects of particulate titanium alloy on the proliferation, differentiation and OPG (osteoprotegerin) gene expression of human osteoblasts in vitro,the purpose was to investigate the mechanism of the periprosthetic osteolysis by wear debris during asepetic loosing. Methods: 1. The authers fetched the embryo calvarial peristeum tissue, got human osteoblast by enzyme-assimilating methods and tissue-block culture methods. We observed the morphological change, growth feature and osteogentic capability, of osteoblast during culture in vitro with phase contrast invert microscope, drew the growth curre and identified the cells by alkaline phosphatase dye. At same time, the morphology and bioactivity of 3-5th-generation osbeoblast and anabiotic cells was studied comparatively. 2. titanium particles were examined by scanning electron and the size was determined by semi-automated image analysis. The 3-5 th gereration of human osteoblast were cultured in medium with different concentration of particulates titanium alloy (1mg/ml, 0.1mg/ml, 0.01mg/ml). Cell growth and proliferation was detected by MTT method after 2,4,6 days that particles were added into medium and ALP ( alkaline phosphatase) activity was measured by kit after 4,7,10 days respectively. 3. With above same methods,the 3-5th generation of human osteoblasts were cultured for 3,6,9days after different concentration of particulates titanium alloy (1mg/ml, 0.1mg/ml, 0.01mg/ml) were added into the medium and OPG(psteoprotegeron) gene expression was quantified by RT-PCR. The statistics of the tests that we mentioned above were analyzed through SARS software. Result: 1.Most of the cultivated periosteal-derived osteoblasts were polygon-like or shullfle-like similar to fibroblast and the 3-5th bgeneration osteoblasts had the stable growth feature and osteogenetic capability. Low temperature effected the surrival rate and proliferation of the osteoblast after cryopreserration. 2. ①The mean size of particle was smaller than 3 μl and it was alike wear debris retrieved from patients seen in vivo.②At the hightest concentration tested (1.0mg/ml)titanium particle reduce cell proliferation(P<0.05), whereas other concentration groups (0.1mg/ml,0.01mg/ml) did not statistically change cell proliferation(P>0.05).③all the different concentration of particle decrease ALP activity (P<0.001)and this effect was not significant at a concentration of 0.1mg/ml and 0.01mg/ml by contrast of 1.0mg/ml. 3. All the different concentration of titanium particles suppressed OPG gene expression of osteoblast. But the effect of particle(0.01mg/ml) on the gene expression decrese gradually over a protacted period of time. Conclusion: ①The cultivated periosteal-derived osteoblast are matured ones which growed and breed rapidly and could become the more ideal seeded cell for studying.The osteoblast after cryperservation was not fit for the research.②Titanium alloy particles reduced cell proliferation and suppress cell osteogenetic capability in a dose-dependent maner.The effect of particles on osteogenetic capability is significant. ③Titanion alloy particles decrease OPG gene expression of human osteoblast.