The Effects Of Calcium And Phosphorus On Proliferation, Differentiation And Mineralization Of SD Rat Osteoblasts In Vitro

In recent years, with the importance of the disease of the bone metabolism, study the effects of different factors on osteoblast (OB) are growing up. OBs to form bone there is to regulate extracellular matrix composition of bone as well as the function of bone matrix mineralization. Therefore, the function status of OB and bone growth to mold, reconstruction and the development of osteoporosis have extremely close ties. Some diseases can increase the number of OBs or enhance the activity of OBs caused an increase in bone mass. Calcium (Ca) and phosphorus (P) are important components of the bone, the process of bone metabolism in the body of animals necessarily involves Ca and P absorption and metabolism. Adequate intake of Ca and P to maintain bone intact animals is essential. The body's Ca balance and Ca metabolism in cells, the concentration of extracellular fluid to maintain the stability of the normal has an important role in bone metabolism. Therefore, to establish a cultural method of fast obtained OBs to study the effect of Ca and P on OBs, there is important theory and guiding significance for the study of osteoporosis.In this study, the experimental parietal bones were obtained from the 4-day-old SD rats, which were subjected to 20min digestion with 2.5 mg/ml trypsin before being cut into fine pieces which were subsequently digested for 1 hour with 1mg/ml collagenase type II to obtain the cell. The cell were identified using alkaline phosphatase (ALP) and calcification nodules staining, trypan blue staining and"repeatedly adherent"cleansing to analysis the survival rate and purity, and using MTT assay with a growth cycle of the activity of OBs distribution, so the model of SD rat parietal bones OBs in vitro is successfully established. OBs were cultured with no added factors (control group), added 1, 2 and 4 mmol/L calcium groups, added 1, 2 and 4 mmol/L phosphorus groups, Ca:P (1:2), i.e. 1 mM Ca and 2 mM P, and Ca:P (2:1), i.e. 2 mM Ca and 1 mM P. We observed morphous, surface structure and fine structure of OBs, measured the proliferation of OBs, ALP activity, content of osteopontin (OPN) and type I collagen (Col- I) and the corresponding mRNA and bone glaprotein (BGP) mRNA transcriptions. Comparing with control group, we found the following: (1) In this study,the cell that we obtained from parietal bones was OBs by identifying with ALP and calcification nodules staining, which purity was reach 98.06%. The survival rate of origin generation OBs was 88.69%, passage OBs was 94.12% by analysis of accounts with trypan blue staining. In a growth cycle, OBs entered into the logarithmic phase on 3-4 day, cells fusion and overlap growth, and reached peak amplitude on day 7, then entered into the calcification phase followed by cells entering the decay phase. Therefore, OBs were treated with Ca and P in cell fusion, to detect variety of indicators on 2nd, 5th and 8th day after treatment with Ca and P. (2) Compared with the control group, Ca in each group and Ca:P (1:2, 2:1) promoted proliferation of OBs, and there is a dose-effects in Ca groups, Ca at 4 mM showed significant effect(P<0.05)on early 2nd day. Ca at 1 and 2 mM showed significant effect(P<0.05)on early 5th day, Ca:P (1:2, 2:1) demonstrated significant effect only on 8th day(P<0.05). P on the proliferation of OBs was not obvious. (3) Compared with the control group, Ca could make the cell body become fuller, needle-like protrusions increased on the surface. The cell morph, architectonic, cell envelope, nuclear membrane was integrity, and mitochondria were mild swelled. P and Ca:P (1:2, 2:1) made the cell body become applanation, needle-like protrusions decreased on the surface. The cell morph, architectonic, cell envelope, nuclear membrane was integrity, and mitochondria cristae fragmentation. (4) Compared with the control group, Ca and P all experiment groups inhibited ALP activity(P<0.05)except Ca at 1 mM on 2nd day, and enhanced the transcription of ALP mRNA on 2nd and 8th days(P<0.05) and inhibited transcription of it on 5th day(P<0.01). (5) Compared with the control group, Ca and P all experiment groups except Ca at 1 mM enhanced the excretion of Col-I on 2nd day(P<0.01), but on 5th and 8th day inhibited its excretion(P<0.05)except P at 2, 4 mM and Ca:P (2:1,1:2)on 5th day and P at 4 mM and Ca:P (1:2) on 8th day; All those enhanced its mRNA transcription on 2nd and 8th day(P<0.01), inhibited its mRNA transcription on 5th day(P<0.01). (6) Compared with the control group, Ca and P all experiment groups on 2nd, 5th and 8th day promoted OPN mRNA transcription (P<0.01)and its excretion(P<0.05) except Ca at 1, 2 mM on 2nd day. (7) Compared with the control group, Ca and P all experiment groups on 2nd, 5th and 8th day promoted BGP mRNA transcription (P<0.01). (8) Compared with the control group, Ca and P all experiment groups enhanced OBs calcification in vitro. All those indicated that Ca at 1, 2 and 4 mM and Ca∶P(1∶2,2∶1)enhanced OBs proliferation, differentiation and calcification in vitro, benefited new bone form. P enhanced OBs differentiation and calcification in vitro, but show not significant effects on OB proliferation.