| Objectives: Lanthanum nitrate was selected as the research object to study its effects on bone marrow mesenchymal stem cells(BMMSC),bone marrow monocytes(BMM)and human umbilical vein endothelial cells(HUVEC)in the bone remodeling process and analyze the potential molecular mechanism of lanthanum nitrate on bone,which provides a theoretical basis for its application in clinical treatment.Methods:(1)Experimental group: Cell and animal experiments were divided into five groups according to the concentration of 0.0001 μmol/L,0.001 μmol/L,0.01 μmol/L,0.1μmol/L and 1 μmol/L lanthanum nitrate,and control group was added with normal saline.(2)Cell viability assay: CCK-8 and Calcein-AM/PI double staining methods were used to determine the effects of different concentrations of lanthanum nitrate on BMMSC and BMM activities of ICR mice.MTT method was used to determine the effects of different concentrations of lanthanum nitrate on HUVEC activity.(3)Mouse BMMSC osteoblasts were cultured in vitro to determine the effects of different concentrations of lanthanum nitrate on osteoblasts differentiation ability by Skeleton Actin staining,alarin red staining,ALP activity staining and q RT-PCR of expression of Bmp2,Runx2,Alp,Ocn,Opg,Rankl,Vegf and other osteoblasts gene.(4)The osteoclast differentiation model of BMM mice was established in vitro to determine the bone erosional capacity of RANKL and M-CSF induced osteoclasts by TRAP staining and bone lacunae test model.(5)The model of angiogenesis in vitro was established,and the effect of different concentrations of lanthanum nitrate on lumen formation ability of HUVEC was determined by the vascularization assay of Matrigel.(6)The osteoporotic culture model of mouse cranial bone was established in vitro and non-direct contact co-culture system model of BMMSC-HUVEC was established in Transwell lab.The protective effect of lanthanum nitrate on bone injury was further explored through quantitative analysis of bone volume fraction,porosity,and angiogenesis results.Results:(1)The results of CCK-8,MTT and Calcein-AM/PI double staining showed that after 1 and 3 days of culture,the proliferation of BMMSC,BMM and HUVEC in different concentrations of lanthanum nitrate was not significantly inhibited,and the results were not statistically significant(P>0.05),suggesting that lanthanum nitrate with preset concentration gradient had no toxic effect on cells.(2)BMMSC was treated with different concentrations of lanthanum nitrate for 7 days.ALP staining and quantitative analysis showed that 0.01 μmol/L lanthanum nitrate could promote the early differentiation of MSC.The results of cytoskeleton Actin staining showed that the cells in 0.0001 and 0.01 μmol/L lanthanum nitrate groups migrated and aggregated significantly,and the spread area of cells was relatively larger than that in the control group.After the intervention of BMMSC with different concentrations of lanthanum nitrate for 21 days,alizer red staining and quantitative analysis results showed that the rate of mineralized nodules in 1μmol /L lanthanum nitrate group was statistically significant compared with the control group(P<0.05),there was no statistical difference among other groups(P>0.05),suggesting that 1μmol/L lanthanum nitrate treatment can promote the late mineralization of BMMSC.(3)After 7 days of routine culture of BMMSC treated with different concentrations of lanthanum nitrate,q RT-PCR analysis of osteoblast-related genes showed that lanthanum nitrate treatment could promote Bmp2 and its downstream Runx2,Ocn and Alp expression,up-regulate Opg level,and inhibit Rankl expression,while had no significant effect on Vegf.(4)The effect of lanthanum nitrate on BMM was investigated by TRAP activity detection and quantitative analysis results.RANKL and M-CSF could effectively induce BMM to differentiate into mature multinucleated osteoclasts,and 0.01 and 1 μmol/L lanthanum nitrate could significantly inhibit the formation of osteoclast and bone resorptive capacity.Further in vitro culture model of mouse skull showed that the porosity and bone volume fraction of the mouse skull were significantly improved after 14 days of culture with the above concentration lanthanum nitrate(P<0.05).(5)Through the establishment of in vitro angiogenesis model,evaluation of 0.0001,0.01,1 μmol/L lanthanum nitrate to the influence of the sample cell lumen structure formation,found that each group is obviously promoted HUVEC generating tube district area and branch number,blood vessel length also increases,which results in the coculture system of BMMSC-HUVEC were also verified,lanthanum nitrate may promote the angiogenesis of HUVEC by acting on BMMSC.Conclusions: The main findings of this study are as follows:(1)Lanthanum nitrate in the concentration range of 0.0001-1 μmol/L has no obvious toxic effects on bone metabolismrelated cells;(2)Lanthanum nitrate can promote the differentiation and maturation of MSC to a certain extent;(3)Lanthanum nitrate concentrations of 0.01 and 1 μmol/L could inhibit the bone resorption activity of osteoclasts by up-regulating OPG/RANKL ratio;(4)Lanthanum nitrate at concentrations of 0.0001,0.01 and 1 μmol/L can promote the angiogenesis of endothelial cells to some extent.(5)Lanthanum nitrate can promote bone repair in osteoblastosteoclast co-culture system and angiogenesis in osteoblast-vascular endothelial cell co-culture system.In conclusion,lanthanum ion compounds have beneficial effects on promoting bone metabolism and improving bone formation. |
