Collagen Peptide Chelating Calcium Effect On Differentiation And Proliferation Of Osteoclasts In Vitro

With osteoclast differentiation factor RANKL(50 ng/ml,72 h)induced RAW264.7(mice macrophage)generated osteoclasts without impurities cells that is good uniformity and has the typical morphological features and function features:Cell body big,the edge is not neat,multi-core,pseudopodia and filamentous swelled,vacuoles can be seen in the cytoplasm,nuclear irregular distribute in the cell;tartrate resistant acid phosphatase staining positive and have lacunae on sclerite.From RAW264.7 cell growth curve induced RANKL can be seen to differentiate in the process of 1d?2d with no cell proliferation;in the process of 2d to 3d cells have both differentiation and proliferation,osteoclasts differentiate into mature and proliferation;in the process of 3d?5d osteoclasts fast proliferate phase;in the process of 5d?9d osteoclasts apoptosis phase.Adding different concentrations of test sample and RANKL to medium of RAW264.7 cell,on the fifth day were determined by MTT,so as to determine the optimum concentration of test sample,and optimum concentration of test sample effect on differentiation,proliferation and apoptosis of osteoclasts.The results show that 17-? estradiol(10?g/ml)and soy isoflavones(100 ?g/ml)compared with blank control significant difference(P<0.05)respectively in 3d?7d?3d?6d of optical density shows that both could significantly inhibit the differentiation and proliferation of osteoclasts,and could significantly promote the apoptosis of osteoclasts.Calcitonin(830 ng/ml)could significantly inhibit the differentiation and proliferation of osteoclasts,parathyroid hormone(100 ?g/ml)were significantly inhibit proliferation of osteoclasts.Sheep bone collagen peptide and calcium chelated sheep bone collagen peptide(high and low in calcium)was 100?g/ml in the optimal concentration,sheep bone marrow peptide and Calcium chelated sheep bone marrow peptide(high and low in calcium)was 1000?g/ml in the optimal concentration,all inhibitory effect on the differentiation of osteoclasts,but the effect is not significant,all significantly inhibit proliferation of osteoclasts,all compared with blank control significant difference(P<0.05)in 4d-5d of optical density.Without the hydrolyzed collagen and negative control BSA have no effect on differentiation,proliferation and apoptosis of osteoclasts.Calcium ions is significant inhibitory effect on proliferation of osteoclasts.Adding the optimal concentration sample and RANKL to RAW264.7 cell culture five days,cell lysis liquid detect of tartrate resistant acid phosphatase(TRAP)activities,the results show that compared with the blank control group,17-? estradiol,soy isoflavones and calcitonin significantly inhibit activity of TRAP on osteoclasts(p<0.01),parathyroid hormone inhibit significantly(p<0.05).Tested sample all inhibit activity of TRAP on osteoclasts.in which calcium chelate peptide(low in calcium)extremely significantly inhibit activity of TRAP on osteoclasts(p<0.01),calcium chelate peptide(high in calcium)is significantly low compared with blank control(p<0.05).Collagen peptide,bone marrow peptide,collagen,BSA,CaCl2 group had no significant difference compared with blank control group.So that only calcium chelate peptide can significantly inhibit the activity of TRAP on the osteoclasts,and the inhibitory effect of low calcium group is higher than the high calcium group.Calcium chelate peptide(low in calcium)and sclerite is co-culture,the results show that the number of lacunae in bone resorption is 34±3.65 by calcium chelated sheep bone marrow peptide(low calcium).The number of lacunae in bone resorption is 32.5±3.69 by calcium chelated sheep bone collagen peptide(low calcium).The number of lacunae in bone resorption is 28.5±2.21 by 17-? estradiol.all significantly lower than the blank control group(43±3.41)(P<0.05).Calcium chelated sheep bone collagen peptide and calcium chelated sheep bone marrw peptide effect on culture of osteoclast in vitro.Its action is similar to the 17-? estradiol,soy isoflavones,calcitonin,and parathyroid hormone,all can significantly inhibit proliferation and bone resorption of osteoclasts,and low calcium content of calcium peptide chelate to TRAP activity inhibition exceed the high calcium content of calcium peptide chelate.Four kinds of hormone,17-?estradiol,soy isoflavones and calcitonin can inhibit differentiation of osteoclasts,17-? estradiol,soy isoflavones also can promote apoptosis of osteoclasts.