Analysis of gene expression during mineralization of cultured human periodontal ligament cells

PURPOSE: Under different culture conditions, periodontal ligament (PDL) stem cells are capable of differentiating into cementoblast-like cells, adipocytes, and collagen-forming cells. Several previous studies reported that because of the stem cells in the PDL, the PDL have a regenerative capacity which, when appropriately triggered, participates in restoring connective tissues and mineralized tissues. Therefore, this study analyzed the genes involved in mineralization during differentiation of human PDL (hPDL) cells, and searched for candidate genes possibly associated with the mineralization of hPDL cells. METHODS: To analyze the gene expression pattern of hPDL cells during differentiation, the hPDL cells were cultured in two conditions, with or without osteogenic cocktails (beta-glycerophosphate, ascorbic acid and dexamethasone), and a DNA microarray analysis of the cells cultured on days 7 and 14 was performed. Reverse transcription-polymerase chain reaction was performed to validate the DNA microarray data. RESULTS: The up-regulated genes on day 7 by hPDL cells cultured in osteogenic medium were thought to be associated with calcium/iron/metal ion binding or homeostasis (PDE1A, HFE and PCDH9) and cell viability (PCDH9), and the down-regulated genes were thought to be associated with proliferation (PHGDH and PSAT1). Also, the up-regulated genes on day 14 by hPDL cells cultured in osteogenic medium were thought to be associated with apoptosis, angiogenesis (ANGPTL4 and FOXO1A), and adipogenesis (ANGPTL4 and SEC14L2), and the down-regulated genes were thought to be associated with cell migration (SLC16A4). CONCLUSIONS: This study suggests that when appropriately triggered, the stem cells in the hPDL differentiate into osteoblasts/cementoblasts, and the genes related to calcium binding (PDE1A and PCDH9), which were strongly expressed at the stage of matrix maturation, may be associated with differentiation of the hPDL cells into osteoblasts/cementoblasts.

Analysis of gene expression during mineralization of cultured human periodontal ligament cells

PURPOSE: Under different culture conditions, periodontal ligament (PDL) stem cells are capable of differentiating into cementoblast-like cells, adipocytes, and collagen-forming cells. Several previous studies reported that because of the stem cells in the PDL, the PDL have a regenerative capacity which, when appropriately triggered, participates in restoring connective tissues and mineralized tissues. Therefore, this study analyzed the genes involved in mineralization during differentiation of human PDL (hPDL) cells, and searched for candidate genes possibly associated with the mineralization of hPDL cells. METHODS: To analyze the gene expression pattern of hPDL cells during differentiation, the hPDL cells were cultured in two conditions, with or without osteogenic cocktails (beta-glycerophosphate, ascorbic acid and dexamethasone), and a DNA microarray analysis of the cells cultured on days 7 and 14 was performed. Reverse transcription-polymerase chain reaction was performed to validate the DNA microarray data. RESULTS: The up-regulated genes on day 7 by hPDL cells cultured in osteogenic medium were thought to be associated with calcium/iron/metal ion binding or homeostasis (PDE1A, HFE and PCDH9) and cell viability (PCDH9), and the down-regulated genes were thought to be associated with proliferation (PHGDH and PSAT1). Also, the up-regulated genes on day 14 by hPDL cells cultured in osteogenic medium were thought to be associated with apoptosis, angiogenesis (ANGPTL4 and FOXO1A), and adipogenesis (ANGPTL4 and SEC14L2), and the down-regulated genes were thought to be associated with cell migration (SLC16A4). CONCLUSIONS: This study suggests that when appropriately triggered, the stem cells in the hPDL differentiate into osteoblasts/cementoblasts, and the genes related to calcium binding (PDE1A and PCDH9), which were strongly expressed at the stage of matrix maturation, may be associated with differentiation of the hPDL cells into osteoblasts/cementoblasts.

The Effects of 1,25-Dihydroxyvitamin D3 on Expression of IGF-I Gene and Cellular Proliferation in MC3T3-E1 Cells / 대한치주과학회지

Polypeptide growth factor belong to a class of potent biologic mediator which regulate cell differentiation, proliferation, migration and metabolism. 1,25-dihydroxyvitamin D3 decrease cell proliferation, and stimulate alkaline phosphatase activity which express in osteoblast during cell differentiation period. IGF-I is known to stimulate cell proliferation and differentiation too. 1,25-dihydroxyvitamin D3 is known to increase IGF-I binding sites and IGF binding protein which inhibite the effect of IGF. The purpose of this study is to evaluate potential role of IGF-I as mediator that control the action of 1,25-dihydroxyvitamin D3. MC3T3-E1 cell were seeded 5x10(5)/ml at 100mm culture plate in alpha-MEM containing 10% fetal bovine serum. After 48 hour incubation period, medium were changed alpha-MEM containing 5% fetal bovine serum. After 24 hours, 10(-9)M 1,25-dihydroxyvitamin D3 added. Total mRNA was extracted at 0, 6, 24, 48, 72 hour. PR-PCR method was programed for the detection of IGF-I mRNA. In the both groups of 1,25-dihydroxy vitamin D3 treated and control, alternative splicing form of IGF-I, IGF-IA and IGF-IB were expressed. In the 1,25-dihydroxyvitamin D3 treated group, IGF-I mRNA expression was matained until 24 hour, there after expression was decresed. MC3T3-E1 cell were seeded 2.5x10(4)/ml at 24well plate in alpha-MEM containing 10% fetal bovine serum. After 48 hour incubation period, medium were changed alpha-MEM containing 3% fetal bovine serum. After 24 hours, 10(-9)M 1,25-dihydroxyvitamin D3 and 10 ng/ml IGF-I were added separately or together. Cell were cultured for 1 and 3 days, 2micronCi/ml [3H]-thymidine was added for the last 24h of culture of each days. [3H]-thymidine incorporation in to DNA was measured and expressed counter per minute(CPM). DNA synthetic activity was significantly decreased by 1,25-dihydroxyvitamin D3 both at 1 day and 3 day, and in the combination group of 1,25-dihydroxyvitamin D3 and IGF-I, DNA synthetic activity was also decreased both at 1 day and 3 days. IGF-I did not affect the DNA synthetic activity compared to control group both at 1 day and 3 day. From the above results, 1,25-dihydroxyvitamin D3 was potent inhibitor of cell proliferaton in MC3T3-E1 cells. It assumed that the effect of 1,25-dihydroxyvitamin D3 on osteoblast proliferation may be mediated in part by decreased level of IGF-I.