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Effects of Icariin on the Proliferation and Osteogenic Differentiation in Human Periodontal Ligament Cells

Author: ZhaoZuoZhou
Tutor: YuZhanHai
School: Lanzhou University
Course: Clinical Stomatology
Keywords: Icariin Human periodontal ligament cells primary culture Proliferation Osteogenic differentiation alkaline phosphatase bone sialoprotein osteocalcin
CLC: R285.5
Type: Master's thesis
Year: 2011
Downloads: 36
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Abstract


Objective:Human periodontal ligament cells (hPDLCs) were primary cultured in vitro and treated with mineralization induced medium containing different concentrations of icariin(ICA). The proliferation of Human periodontal ligament cells, alkaline phosphatase (ALP) activity, bone sialoprotein (BSP) and osteocalcin (OCN) were performed to assess the effects of icariin on the proliferation and osteogenic differentiation.Methods:1.Human periodontal ligament cells(hPDLCs) were primary cultured in vitro by the use of enzyme digestion combine coverslip covering tissue culture. The morphological characteristics were observed with an invert microscope and growth curves were drew by using daily cell counting.2.The human periodontal ligament cells were identified with vimentin and cytokeratin immunohistochemical staining.3. Under the conditions of mineralization induce, human periodontal ligament cells were cultured 21 days. Then alizarin red staining was used and calcified nodules were observed to evaluate the cells ability of osteogenic differentiation.4. Under the conditions of mineralization induce,10μg/ml、1μg/ml、0.1μg/ml、0.01μg/ml、0.001μg/ml、Oμg/ml icariin medium were added to human periodontal ligament cells. MTT colorimetric assay was used to observe the proliferation of human periodontal ligament cells at 24th、48th、72th hours. To assess the influence of icariin on the proliferation of human periodontal ligament cells.5. Under the conditions of mineralization induce,10μg/ml、1μg/ml、0.1μg/ml、0.01μg/ml、0.001μg/ml、0μg/ml icariin medium were added to human periodontal ligament cells. After 72 hours, alkaline phosphatase (ALP) activity, bone sialoprotein (BSP) and osteocalcin (OCN) were observed to investigate the influence of icariin on the osteogenic differentiation of human periodontal ligament cells.Results:1.Through enzyme digestion combine coverslip covering tissue culture, human periodontal ligament cells(hPDLCs) can be cultured successfully and growth curves can be seen. The number of cells decreased after inoculating 24-48 hours. However, the number of cells begin to increase at 3rd day and graduate into exponential proliferative stage. And the number of cells reached maximum at 7th day, then dropped down to platform period from 8th day. The growth curve was similar to "S" shape. According to a formula, the cells multiplication time is about 90 hours.2.The human periodontal ligament cells were identified with immunohistochemical staining. The results showed that the expression of vimentin was positive and cytoplasm’s stain was brown. The expression of cytokeratin was negative and there was no stain in cytoplasm. All of these proved that the cells derived from mesoblast.3. Under the conditions of mineralization induce, grey nodules can be found in the human periodontal ligament cells and they increased in size and number over time. The cells were cultured 21 days. Then alizarin red staining was used and many red stained calcified nodules were observed in the mineralization induced group. While there was few red stained calcified nodules in the control group. The results suggest human periodontal ligament cells have the ability of osteogenic differentiation.4.The influence of icariin on the proliferation of human periodontal ligament cells All of the concentrations of icariin promoted proliferation of human periodontal ligament cells for 24 hours(P<0.05).1～0.001μg/ml icariin promoted proliferation of human periodontal ligament cells for 48 hours (P<0.05). Compared with the control group, OD value was small in 10μg/ml group. But the difference was not significant (P>0.05).0.1～0.001μg/ml icariin promoted proliferation of human periodontal ligament cells for 72 hours (P<0.05),while 10μg/ml icariin inhibited proliferation of human periodontal ligament cells (P<0.05)5.The influence of icariin on the alkaline phosphatase (ALP) activity of human periodontal ligament cells Under the conditions of mineralization induce,1～0.001μg/ml icariin improved ALP activity expression for 72 hours (P<0.05). While there were no significant difference between 10μg/ml group and control group (P>0.05)6.The influence of icariin on the expression of bone sialoprotein (BSP) of human periodontal ligament cells Under the conditions of mineralization induce, compared with the control group, the bone sialoprotein (BSP) expression of 0.1～0.001μg/ml cariin were increased significantly for 72 hours (P<0.05). However, the expression of 1 u g/ml group and 10μg/ml group were similar to the control group (P>0.05)7. The influence of icariin on the expression of osteocalcin (OCN) of human periodontal ligament cells Under the conditions of mineralization induce, osteocalcin (OCN) expression of 1～0.01μg/ml cariin were increased significantly for 72 hours compared with the control group (P<0.05). The group of 0.001μg/ml showed higher osteocalcin (OCN) expression level than the control group, but there were no difference between the two groups (P>0.05). While 10μg/ml icariin inhibited OCN expression of human periodontal ligament cells and had statistical signification (P<0.05)Conclusion:1.Enzyme digestion combine coverslip covering tissue culture has a short culture period, high successful rate, reliable origin and good active condition. So it can be regarded as an successful method for human periodontal ligament cells(hPDLCs) culture.2.Cell cluster of human periodontal ligament cells(hPDLCs) have the ability of osteogenic differentiation.3.Appropriate icariin could promote proliferation and osteogenic differentiation in human periodontal ligament cells.

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