The Effect Of Nicotine On The Proliferation And Osteogenic Differentiation Of Rabbit Marrow-origin Osteoblasts

Objective: Through studying the effects of nicotine on the proliferation and osteogenic differentiation of rabbit marrow-origin osteoblasts (MOOBs) in vitro, to learn the action mechanism of tabacco on periodontitis, peri-implantitis and implant osseointegration.Method: The BMSCs of rabbit were isolated and cultured in vitro.The 3rd generation BMSCs were induced in osteogenic media. After 2 weeks culture, differentiating BMSCs (MOOBs) were treated with nicotine at the concentrations of 0.01,0.1,1,10,100μg/ml, and compared with the control group only in ostogenic media. The growth OD values were measured by MTT colorimetric assay on the 1st, 3rd, 5th and 7th day to estimate the proliferative ability of MOOBs. The alkaline phosphatase (ALP) activity values were measured on the 1st, 3rd, 5th and 7th day to estimate the osteogenic potential of MOOBs.Results: 1. In this experiment, BMSCs cultured with 10nmol/L dexamethasone, 10mmol/Lβ-glycerophosphate sodium, 50mg/L L-ascorbic acid were induced osteoblast differentiation, and gradually became short spindle. ALP staining of MOOBs showed alkaline phosphatase was positive. Von kossa staining showed there were calcium noduses in cell-intensive areas.2. After the treatment with different concentrations of nicotine, the groups of below 10ug/ml didn't appear to be obviously inhibitory not only in cell multiplication but in ALP activity compared to the control groups (P>0.05).The group of 10ug/ml on the 5th,7th day and 100ug/ml group showed inhibitory action to the cell multiplication(P< 0.05). The group of 10ug/ml on the 3rd, 5th,7th day and 100ug/ml group could reduce ALP activity of MOOBs, and the differences between experiment groups and control groups were statistically significant (P<0.05). Nicotine in higher concentrations could significantly inhibit MOOBs proliferation and osteogenic differentiation in a dose-dependent and a time-dependent manner.Conclusion: 1. In this experiment, BMSCs of rabbits under specific osteogenic conditions can be induced osteoblast differentiation, and to be bone marrow stromal cells (MOOBs) for the vitro study.2. The concentration range of the plasma level of nicotine attained in the smokers didn't appear to be obviously inhibitory not only in cell multiplication but in ALP activity in this experiment, and higher concentrations caused dose-dependent inhibitory effects. Considering the high levels of nicotine attained in the crevicular fluid and saliva of tobacco users and the high lipophilic and diffusible character of this substance, results suggest nicotine, the important toxin in tobacco, may play local pathological role on periodontitis, peri-implantitis and osseointegration, but not through systemic effects. Meanwhile, due to low concentrations of nicotine didn't caused inhibitory effects to MOOBs, we should realize that, because there are many different toxins and various influence factors in the smog, the negative impact of smoking on dental implantation and periodontitis may be related to more than one molecule present in the cigarette smoke, and nicotine seems to be only one promoting agent.