Study of human apical papilla cells cultured on amniotic membrane matrix
Date Issued
2009
Date
2009
Author(s)
Chung, Min-Chun
Abstract
Human amniotic membrane (AM) is a nature biomaterial which has been successfully used in cornea reconstruction and as a biological dressing for wounds. Recently, it was reported that apical papilla cells were multipotent and their ability of differentiating into specific lineage could be induced in an appropriate environment. Therefore, the purpose of this study was to investigate the possibility of applying human amnions for oral tissue repair by evaluating the growth and function of human apical papilla cells cultured in the de-cellular human amniotic matrix. In addition to apical papilla cells, several kinds of dental mesenchymal cells were used to check the feasibility of de-cellular AM matrix for cellular adhesion and growth, including dental pulp cells, gingival fibroblasts, periodontal ligament fibroblasts, and bone marrow mesenchymal stem cells. The apical papilla cells were cultured from human apical papilla of developing third molar extracted due to orthodontic reason. The dental progenitor cells in the mixed population cultured from apical papilla was characterized by using anti- STRO-1 and anti- CD146 antibody for immunofluorescence stain and flow cytometry. The osteogenic differentiation of apical papilla cells grown on different substrates (tissue culture plate (TCPS), basement membrane of AM, and collagenous matrix of AM) was investigated by measuring the alkaline phosphotase activity and extra-cellular matrix mineralization revealed by von Kossa stain. The mRNA expressions of osteoblast-related genes (COL I, Cbfa1, ALP, and OC) were checked by using RT-PCR technique. Protein expression and phosphorylation of RUNX-2 were detected via the procedures of imunoprecipitation and western blot. Moreover, we used U0126, an inhibitor of ERK1/2 pathway, to check if the ERK 1/2 pathway is involved in the osteogenic differentiation of apical papilla cells grown on AM. The result showed that the adhesion and proliferation of apical papilla cells cultured on amniotic membrane were not as good as those on TCPS. The similar difference between amniotic membrane and TCPS for cellular growth was noted for dental pulp cells and bone marrow mesenchymal stem cells. However, amniotic membrane was a good matrix to support the growth of gingival fibroblasts and periodontal ligament fibroblasts. The osteoblastic phenotype of human apical papilla cells was demonstrated by their progressive increase of ALP activity and extracellular matrix mineralization in long-term culture. Compared to TCPS, AM could promote osteogenic differentiation of apical papilla cells. The mRNA expression of COL I, Cbfa1, ALP, and OC was also up-regulated in apical papilla cells grown on AM. Moreover, the osteogenic differentiation of apical papilla cells, either on TCPS or on AM, was significantly down-regulated in the presence of U0126. It implied that ERK1/2 signaling pathway was activated in the osteogenic differentiation of apical papilla cells, followed by up-regulating the expression and phosphorylation of RUNX-2, subsequently increasing ALP activity and mineralized matrix deposition. However, the promoting effect of AM on the osteogenic differentiation of apical papilla cells may not directly involve the ERK1/2 signaling pathway.
Subjects
human amniotic membrane
apical papilla cells
osteogenic differentiation
Type
thesis
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