Study About Effect Of Collagen Peptide Calcium Chelating On Osteoblast Proliferation And Activity

The study is aimed to investigate the effect of bone collagen polypeptide(CP),bone marrow polypeptide(MP)polypeptides and their calcium-chelated form,CCCP and CCMP.on the proliferation and activity of cultured osteoblasts.The comparison were also conducted with the estrogen.The paper describes the principle of improving the bone,lay the foundation for the development of calcium preparations.The main results are as follows:Skulls of Neotal rats(within 24h)were digested by trypsinase and type-II collagenase sequentially or simultaneously to prepare the osteoblast(OB).OBs with high purity and good stability were obtained after culturing 3 generations in DMEM/F12 whole medium.The resulted OBs showed the typical cyto-morphologic and functional features,they were positively stained after alkaline phosphatase staining and alizarine red staining.The growth curve were made by CCK-8 methodology and Day 2?5 was the exponential period of OB cell.All the tested agents with different concentrations were added to the cell culture on Day 2 and the OD value were measured on Day 5,thus determine the optimal concentration of each agents in later experiments.After determining the functions on the OB's growth of polypeptide and calcium-chelated peptides with the optimal concentration.The resulted suggested that CP and CCCP at the concentration of 102?g·mL-1,MP and CCMP with the concentration of 103?g·mL-1?and all positive controls,namely soybean isoflavone with the concentration of 10?g·mL-1,E2,PTH and CAT with the concentration of 8.3×10-1ng·mL-1,in contrast with blank control group,can significantly promote the OB proliferation in 1?7 day(P<0.05).The result shows that all of them can promote the proliferation of osteoblasts.ALP is an important symbolic molecule of cultured osteoblast's activity.The resulted suggested that the activity of all positive control(E2?soybean isoflavone?CAT)groups is significantly higher than the blank control group(P<0.01).All calcium-chelated peptides(CCCP and CCMP with high Ca and low Ca)Can significantly promote osteoblast to secrete ALP,and the low Ca groups are superior than the high Ca groups.Compared with the blank control group,the CCMP and CCCP with low Ca content works extraordinarily(P<0.01),the CCMP and CCCP with higf Ca content works(P<0.05).Polypeptide(CP and MP)n BSA?non-hydrolyzed bone collagen?PTH and CaC12 showed no effects on the ALP activity of OB(P>0.05).BGP is a symbolic molecule of OB in medium phase of differentiation which is essential for bone calcification.The resulted suggested that all of 4 calcium-chelated peptides and all positive controls can significantly improve the BGP level of OB(P<0.05),among them CCMP with high Ca content works extraordinarily(P<0.01).Bone collagen also increased the BGP level((P<0.05))when compared to the blank control.While CP,MP,CaC12 and BSA showed no effect.In summary,CP,MP and free Ca2+ could enhance the proliferation of OB,but showed no effect on the activity of OB.Like 17 ?-estradiol,CCCP and CCMP,can enhance both the proliferation and the activity of OB.While BSA,non-hydrolyzed bone collagen showed no effects on the proliferation or activity of OB.Therefore calcium-chelated polypeptides had the estrogen-like functions on the proliferation and activity of OB.