Amelogenin（AMG） is a cell adhesion molecule that has an important role in the mineralization of enamel and regulates events during dental development and root formation. The purpose of the present study was to inves...Amelogenin（AMG） is a cell adhesion molecule that has an important role in the mineralization of enamel and regulates events during dental development and root formation. The purpose of the present study was to investigate the effects of recombinant human AMG（rhAMG） on mineralized tissue-associated genes in cementoblasts. Immortalized mouse cementoblasts（OCCM-30）were treated with different concentrations（0.1, 1, 10, 100, 1 000, 10 000, 100 000 ng · mL^-1） of recombinant human AMG（rhAMG）and analyzed for proliferation, mineralization and mRNA expression of bone sialoprotein（BSP）, osteocalcin（OCN）, collagen type I（COL I）, osteopontin（OPN）, runt-related transcription factor 2（Runx2）, cementum attachment protein（CAP）, and alkaline phosphatase（ALP） genes using quantitative RT-PCR. The dose response of rhAMG was evaluated using a real-time cell analyzer.Total RNA was isolated on day 3, and cell mineralization was assessed using von Kossa staining on day 8. COL I, OPN and lysosomalassociated membrane protein-1（LAMP-1）, which is a cell surface binding site for amelogenin, were evaluated using immunocytochemistry. F-actin bundles were imaged using confocal microscopy. rhAMG at a concentration of 100 000 ng · mL^-1 increased cell proliferation after 72 h compared to the other concentrations and the untreated control group. rhAMG（100 000 ng · mL^-1） upregulated BSP and OCN mRNA expression levels eightfold and fivefold, respectively. rhAMG at a concentration of 100 000 ng · mL^-1 remarkably enhanced LAMP-1 staining in cementoblasts. Increased numbers of mineralized nodules were observed at concentrations of 10 000 and 100 000 ng · mL^-1 rhAMG. The present data suggest that rhAMG is a potent regulator of gene expression in cementoblasts and support the potential application of rhAMG in therapies aimed at fast regeneration of damaged periodontal tissue.展开更多
The interaction of proteins with solid surfaces is a fundamental phenomenon in the biomaterials field. We investigated, using atomic force microscopy (AFM), the interactions of a recombinant amelogenin with titanium, ...The interaction of proteins with solid surfaces is a fundamental phenomenon in the biomaterials field. We investigated, using atomic force microscopy (AFM), the interactions of a recombinant amelogenin with titanium, a biphasic calcium phosphate (BCP) and mica. The unbinding processes were compared to those of an earlier studied protein, namely fibrinogen. Force spectroscopy (AFM) experiments were carried out at 0 ms, 102 ms, 103 ms and 104 ms of contact time. In general, the rupture forces increased as a function of interaction time. The unbinding forces of amelogenin interacting with the BCP surface were always stronger than those of the amelogenin-titanium system. The unbinding forces of fibrinogen interacting with the BCP surface were always much stronger than those of the fibrinogen-titanium system. For the most part, this study provides direct evidence that recombinant amelogenin binds more strongly than fibrinogen on the studied substrates.展开更多
基金supported by TüBITAK SBAG108S265(Turkey)BMBF TUR08/09(Germany)
文摘Amelogenin（AMG） is a cell adhesion molecule that has an important role in the mineralization of enamel and regulates events during dental development and root formation. The purpose of the present study was to investigate the effects of recombinant human AMG（rhAMG） on mineralized tissue-associated genes in cementoblasts. Immortalized mouse cementoblasts（OCCM-30）were treated with different concentrations（0.1, 1, 10, 100, 1 000, 10 000, 100 000 ng · mL^-1） of recombinant human AMG（rhAMG）and analyzed for proliferation, mineralization and mRNA expression of bone sialoprotein（BSP）, osteocalcin（OCN）, collagen type I（COL I）, osteopontin（OPN）, runt-related transcription factor 2（Runx2）, cementum attachment protein（CAP）, and alkaline phosphatase（ALP） genes using quantitative RT-PCR. The dose response of rhAMG was evaluated using a real-time cell analyzer.Total RNA was isolated on day 3, and cell mineralization was assessed using von Kossa staining on day 8. COL I, OPN and lysosomalassociated membrane protein-1（LAMP-1）, which is a cell surface binding site for amelogenin, were evaluated using immunocytochemistry. F-actin bundles were imaged using confocal microscopy. rhAMG at a concentration of 100 000 ng · mL^-1 increased cell proliferation after 72 h compared to the other concentrations and the untreated control group. rhAMG（100 000 ng · mL^-1） upregulated BSP and OCN mRNA expression levels eightfold and fivefold, respectively. rhAMG at a concentration of 100 000 ng · mL^-1 remarkably enhanced LAMP-1 staining in cementoblasts. Increased numbers of mineralized nodules were observed at concentrations of 10 000 and 100 000 ng · mL^-1 rhAMG. The present data suggest that rhAMG is a potent regulator of gene expression in cementoblasts and support the potential application of rhAMG in therapies aimed at fast regeneration of damaged periodontal tissue.
文摘The interaction of proteins with solid surfaces is a fundamental phenomenon in the biomaterials field. We investigated, using atomic force microscopy (AFM), the interactions of a recombinant amelogenin with titanium, a biphasic calcium phosphate (BCP) and mica. The unbinding processes were compared to those of an earlier studied protein, namely fibrinogen. Force spectroscopy (AFM) experiments were carried out at 0 ms, 102 ms, 103 ms and 104 ms of contact time. In general, the rupture forces increased as a function of interaction time. The unbinding forces of amelogenin interacting with the BCP surface were always stronger than those of the amelogenin-titanium system. The unbinding forces of fibrinogen interacting with the BCP surface were always much stronger than those of the fibrinogen-titanium system. For the most part, this study provides direct evidence that recombinant amelogenin binds more strongly than fibrinogen on the studied substrates.
