Study On The Difference Of Osteoblasts From Femur And Skull In C57BL/6 Mice

Objective:To observe the biological characteristics of femoral BMMSCs and skull cells of C57BL/6 mice,verified and compared the osteogenic ability of osteoblasts from femoral and skull of C57BL/6 mice of different ages and the expression levels of hematopoietic related factors CXCL12,IL-6,SCF and N-cadherin in vitro,and explored a simple,rapid and efficient method for osteoblast culture.Methods:1.Femurs of C57BL/6 mice aged 6 weeks and 10 months were obtained,then collecting bone marrow.Culturing and purifing BMMSCs by whole bone marrow adherent method,and observing the BMMSCs of cells under microscope.2.The skulls of C57BL/6 mice aged 6 weeks and 10 months were cut into small bone pieces,then culture the skull cells by tissue block method combined with enzyme digestion method,and observe the cell morphology and growth by the microscope.3.Collecting the third generation BMMSCs and skull cells,and analyzing the surface antigens of the two cells by flow cytometry.4.Osteoblasts were induced by dexamethasone,β-glycerophosphate and vitamin C for 2 weeks,respectively,and identified by alizarin red staining.5.The expression of osteogenic indexes ALP,Runx2 and COL1 genes in BMMSCs and osteoblasts from skull of 6-week-old and 10-month-old mice was detected by qRT-PCR;The expression of Runx2 protein in osteoblasts of 6-week-old mice from two sources was detected by Western blot.6.(1)The expression of hematopoietic factors CXCL12,IL-6,SCF and N-cadherin in BMMSCs and osteoblasts from skull of 6-week-old and 10-month-old mice was detected by qRT-PCR;Detecting the concentrations of CXCL12,IL-6 and SCF in the supernatant of osteoblasts from 6-week-old and 10-month-old mice on the14 th day of culture by ELISA.⑵Detecting the expression of N-cadherin protein in osteoblasts of 6-week-old mice from two sources Western blot.Results:1.Cell morphology: There is little difference between BMMSCs and skull cells.The primary cultured BMMSCs are irregular in shape,such as paving stone,triangle and spindle.After subculture,the cells are basically spindle-shaped,with smaller cell bodies and shorter processes.The primary cultured skull cells grow around the skull fragments in the shape of spindle,triangle,etc.After passage,the skull cells have a uniform long spindle shape,and the cell body of the skull cells is slightly larger than that of BMMSCs,and the process is longer.2.Proliferation: Primary BMMSCs cultured in vitro proliferated faster than primary skull cells.After cell passage,skull cells only need 4～5 days to grow80 %～90% of the dish bottom;After 4 days of BMMSCs culture,the cells are still sparse,and the bottom of the dish is 80 %～90% after about 8～10 days of culture.3.Cell surface markers: BMMSCs and skull cells are highly expressing CD44 and CD29,but hardly expressing CD34 and CD45.4.osteogenic ability:⑴ The results of alizarin red staining were positive after BMMSCs and skull cells were induced into osteoblasts for two weeks.The expression of Runx2,ALP and COL1 m RNA in osteoblasts derived from skull cells of 6-week-old mice were more than that of osteoblasts derived from BMMSCs,and the results of 10-month-old mice were consistent with those of 6-week-old mice.⑵ Osteoblasts from BMMSCS: The expression of ALP m RNA in 10-month group was significantly more than that in 6-week group,and there was no significant difference between the two groups in Runx2 and COL1 m RNA levels.Osteoblasts from skull: The expression of ALP m RNA in 10-month group was significantly more than that in 6-week group,and there was no prominent difference in the relative expression of Runx2 and COL1 m RNA between the two groups.5.Expression of cytokine:⑴ 6-week-old mice: Compared with osteoblasts derived from BMMSCs,the expression level of SCF gene in osteoblasts derived from skull and its concentration in cell culture supernatant decreased;Compared with BMMSCs-derived osteoblasts,VI osteoblasts from skull expressed higher N-cadherin gene and protein.There was no protuberant difference between qRT-PCR and ELISA in detecting CXCL12 and IL-6between the two groups.⑵10-month-old mice: Compared with osteoblasts derived from BMMSCs,the expression level and concentration of SCF gene in osteoblasts derived from skull decreased;Compared with BMMSCs-derived osteoblasts,osteoblasts from skull express more N-cadherin m RNA.There was no prominent difference between qRT-PCR and ELISA in detecting CXCL12 and IL-6 between the two groups.⑶ Osteoblasts from BMMSCs: There was no prominent difference in the m RNA levels of CXC12,IL-6,SCF and N-cadherin and the concentrations of CXC12,IL-6 and SCF in cell culture supernatant between the 6-week group and the 10-month group.⑷ Osteoblasts from skull: There was no prominent difference in the m RNA levels of CXC12,IL-6,SCF and N-cadherin and the concentrations of CXC12,IL-6and SCF in cell culture supernatant between the 6-week group and the 10-month group.Conclusions:1.There is no obvious difference in morphology and cell surface antigen expression between BMMSCs and skull cells,and they are basically spindle-shaped,with high expression of CD44 and CD29,but no expression of CD34 and CD45;2.The proliferation of primary BMMSCs is faster than that of primary skull cells,but the proliferation of skull cells after cell passage is faster than that of BMMSCs;3.BMMSCs and skull cells can differentiate into osteoblasts in vitro,and the osteogenic ability of skull cells is higher than BMMSCs;4.Osteoblasts from BMMSCs expressed SCF higher than those from skulls,but the expression of N-cadherin was lower than that from skulls,and there was no significant difference between CXCL12 and IL-6.5.There is no significant difference in osteogenic differentiation and expression of hematopoietic factors between BMMSCs of young and adult rats and osteoblasts from skull.This experiment proves that osteoblasts from skull are simple to extract,convenient to culture,fast in cell growth and high in osteogenic ability,and they are good in vitro research models of osteoblasts.