| Background/Object:to investigate the osteogenic activity of the Bone marrow mesenchymal stem cells(BMSCs)in nano fiber scaffold,construction of bioactive tissue engineering scaffold KLD-12 peptide/ rhBMP-2 gel combined with BMSCs,by using the release of characteristic of KLD-12 peptide,and combined the appropriate dose of rhBPM-2.Methods: BMSCs were isolated from bone marrow of New Zealand rabbits(aged male and female)in March.The third generation BMSCs respectively by rhBMP-2 / KLD-12 peptide concentration in three-dimensional culture for training(experimental group)and KLD-12peptide(control group),BMSCs induced osteogenesis.cell counting kit-8 assay was used to detect the proliferative activity of the cells in the experimental group and the control group on2,5,7,10,14d;Calcein-AM/ propidium iodide fluorescence staining was observed in living cells and dead cells in the experimental group and the control group at 7d and 14 d,and calculate the ratio of living cells;and the content of alkaline phosphatase(ALP)and Osteocalcin(Osteocalcin,OCN)in the two groups were detected on 3,7,10,14,21d;and the experimental group and the control group were treated with collagen type I immunofluorescence staining at 14d;and the relative expression of collagen type I and OCN was detected by real-time quantitative PCR.Result:(1)Under the inverted microscope,the BMSCs were cultured in circular shape in the two groups at 7d,partial cells proliferation,and the edge of the gel was spindle shaped;(2)CCK-8 results showed that: with prolonged incubation time,cells proliferation activity were increased in the experimental group and the control group,compared with other time points,the proliferation activity was the highest on 14 d,the difference was statistically significant(P< 0.05),except for second days,the proliferative activity of the experimental group was higher than that of the control group at each time point,the difference was statistically significant(P < 0.05);(3)Calcein-AM/PI fluorescence staining showed that there were round viable cells in the gel at 7d and 14 d,and on 14 d the proliferation of cells was more obvious than that of 7d,and there were a few dead cells,and the cell survival rate in the experimental group and the control group were 92.26±2.28%?90.56±3.5%,respectively,in the 14 d,there was no significant difference between the two groups(P > 0.05);(4)The results of ALP detection showed that there was no significant difference in the expression of ALP in two groups in the course of culture for 3d(P > 0.05),and the expression of ALP in the experimental group was significantly higher than that in the control group at 7-21d(P<0.05);(5)the results of OCN test showed that there was no significant difference in OCN content between the two groups at 3d and 7d(P > 0.05).The content of OCN in experimental group was significantly higher than that in control group at 10-21d(P<0.05);(6)the observation of laser confocal microscope showed that: the immunofluorescence staining of Collagen? waspositive in the experimental group,and the fluorescence intensity was stronger than that of the control group after 14 d of culture.(7)The results of qRT-PCR showed that the expression of collagen type I and osteocalcin gene in experimental group was significantly higher than that of control group,and the difference was statistically significant(P<0.05)Conclusion: BMSCs can grow and proliferate in KLD-12 polypeptide/rhBMP-2,and BMSCs was induced to differentiate into osteoblasts of good biological activity in KLD-12 polypeptide /rhBMP-2 gel,specificity of osteogenic marker ALP,OCN and collagen type I expression were increased,lays the foundation for further research on the application of KLD-12 peptide / rhBMP-2/BMSCs gel for treatment of lumbar instability minimally invasive treatment of the new scheme. |
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