Differentiation of human ameloblast‐lineage cells in vitro

Abstract

Previous studies have shown that ameloblast-like cells can be selectively cultured from the enamel organ in a serum-free medium with low calcium concentrations. The purpose of this study was to further characterize this culture system to identify differentiated ameloblast-lineage cells. Tooth organs from 19-24-wk-old fetal cadavers were either frozen and cryosectioned for immunostaining, or digested in collagenase/dispase for cell culture. The cells were grown in keratinocyte media supplemented with 0.05 mM calcium, and characterized by morphology and immunofluorescence. Epithelial clones with two distinct morphologies, including smaller cobblestone-shaped cells and larger (5-15 times in size) rounded cells, began to form between day 8 and day 12 after culture. The cobblestone-shaped cells continued to proliferate in culture, while the larger cells proliferated slowly or not at all. These larger cells formed filopodia, usually had two or more nuclei and a radiating cytoplasm at the cell margin, and were more abundant with increasing time in culture. Both cell types stained for cytokeratin 14, and the larger cells appeared more differentiated, showing stronger staining for amelogenin and ameloblastin. Immunofluorescence of the tooth bud sections showed staining for these matrix proteins as ameloblasts differentiated from the inner enamel epithelium. These results show the successful culture of differentiating ameloblast-lineage cells, and lay a foundation for use of these cells to further understand ameloblast biology with application to tooth enamel tissue engineering.

Calcium‐mediated differentiation of ameloblast lineage cells in vitro

“…Primary ameloblast lineage cells were isolated from 18 to 23-week-old fetal tooth buds, with permission obtained through the tissue-sharing program at UCSF, as previously described (DenBesten et al, 2005;Yan et al, 2006;Zhang et al, 2006;Le et al, 2007;Zhang et al, 2007). Briefly, the tissue mass from these tooth buds was dispersed by the addition of 1 mg/mL collagenase/ dispase mixture dissolved in PBS at 371C for 2 hr.…”

JNK/c-Jun signaling pathway mediates the fluoride-induced down-regulation of MMP-20 in vitro

“…This cell population was further enhanced by removing fibroblast-like cells with STV solution impregnated cloning discs (Scienceware, GA). Selected ameloblast lineage cells were characterized by immunochemical staining with cytokeratin 14, amelogenin and MMP-20 antibodies (Tabata et al, 1996;Yan et al, 2006). For fluoride treated samples, 10 μM NaF was added into the growth medium as cells reached 80% confluence, following overnight synchronization in growth factor-free media (KBM).…”

“…This has been in part due to the lack of suitable ameloblast culture systems. Our laboratory has successfully isolated, cultured and characterized primary ameloblast lineage cells (DenBesten et al, 2005;Yan et al, 2006). In this current study, we investigated the effects of micromolar fluoride on MMP-20 expression and the molecular pathway transmitting a fluoride-induced alteration of MMP-20 in ameloblast lineage cells.…”

Introduction of a three-dimensional and layered (TDL) culture, a novel primary co-culture method for ameloblasts and pulp-derived cells

“…5, 7) appear to be the cells of an ameloblast lineage recapitulating the structural and functional properties of presecretory ameloblasts. Such "non-flat" morphological feature of the enamel epithelial cells has not been demonstrated in previous primary cultures of enamel epithelial cells (Limeback 1987;Kukita et al, 1992;Chen et al, 1992;Tabata et al, 1996bTabata et al, , 2003Matsumura et al, 1998;DenBesten et al, 1998DenBesten et al, , 2005Suzawa et al, 2006;Yan et al, 2006). The cells prepared by the method by Kukita et al (1992) from the epithelial sheet of the rat incisor enamel organ naturally contain various types and functional states of cells of enamel epithelia and have some unavoidable contamination of mesenchymal cells.…”

“…The primary culture of enamel epithelial cells has been attempted by many investigators using enamel organderived cells from porcine (Limeback 1987, DenBesten et al, 1998, mouse (Chen et al, 1992;Suzawa et al, 2006), rat (Kukita et al, 1992;Tabata et al, 1996bTabata et al, , 2003Matsumura et al, 1998) and human (DenBesten et al, 2005;Yan et al, 2006) tooth germs under different conditions. In a primary culture, the ameloblast-lineage cells after inoculation generally form a monolayer sheet of closely packed cells of cobblestone-like profiles.…”