Electron microscopy, micro-analysis, and X-ray diffraction characterization of the mineral-like tissue deposited by human cementum tumor-derived cells.

The nature and characteristics of the mineralized-like tissue deposited by cementoblasts are not well-known due to the difficulties in obtaining and culturing cells representing the cementum phenotype. We hypothesized that a putative cementoblastic cell line derived from a human cementoblastoma could serve as a suitable model to study the physical, chemical, and morphological features of the cementum-like tissue deposited in vitro. The cementoblastoma cell line was studied by transmission electron, high resolution, scanning, and atomic force microscopy and compared with human cellular cementum, human osteoblasts, and human alveolar bone. The analyses of the crystals and mineral-like tissue in the cell line were performed by x-ray diffraction microscopy and energy-dispersive x-ray micro-analysis. TEM examination of cementoblastoma cells revealed the presence of electron-dense intracellular vesicles surrounded by a membrane that contained filaments and needle-like structures. The diffraction patterns obtained from the intracellular material and human cellular cementum were similar, with D-spacings of 3.36 and 2.8, consistent with those of hydroxyapatite (3.440 and 2.814). The composition of the mineral-like tissue had a Ca/P ratio of 1.60 for cementoblastoma cells and 1.97 for human cellular cementum. Na (5.29%) and Cl (1.47%) were present in the composition of cementoblastoma cells. Human cellular cementum additionally contained Mg (4.95%). Osteoblastic cells showed a Ca/P ratio of 1.6280. Na represented 4.52% and Cl 1.22% of its composition. Human alveolar bone had a Ca/P ratio value of 2.01. Na (6.63%), Mg (2.10%), and Cl (0.84%) were also present. All samples examined represented biological-type hydroxyapatite. Based on the compositional and morphological features, these findings indicate that cementoblastoma-derived cells express the human cellular cementum phenotype.

Human cementum tumor cells have different features from human osteoblastic cells in vitro.

Cells obtained from human cementoblastoma and alveolar bone were isolated and cultured. Initial and late stages of mineralization were assessed by using atomic force microscopy, scanning electron microscopy and X-ray microanalysis. In cultures of cementoblastoma-derived cells the initial stages of mineralization showed well-defined spherical-shaped structures, while the osteoblastic cells showed plaque-like deposits. These morphological patterns of mineral deposition could serve as nucleation centers for hydroxyapatite crystals. Late stages of mineralization at 28 and 35 d maintained those morphological differences established in initial cultures. The material deposited by cementoblastoma and osteoblastic cells, analyzed by EDX spectra, revealed similar Ca/P ratios for both cell types. These values were similar to those reported for hydroxyapatite in enamel and bone. Alkaline phosphatase specific activity (AlP), of osteoblastic cells at 3, 7 and 11 d, showed an increase of 27.9, 50.9 and 37.0% (p < 0.001), respectively. However, at 15 and 19 d there was an increase of AlP activity of cementoblastoma cells by 39.4 and 34.5% over osteoblastic cells (p < 0.001). Immunostaining of cementoblastoma and osteoblastic cells using a specific mAb against a cementum-derived attachment protein revealed strong immunostaining of cementoblastoma cells which was localized to the cell membrane and fibril-like structures (96.2 +/- 1.3). A few osteoblastic cells also stained weakly with the anti-CAP mAb (6.4 +/- 0.6). Sections of decalcified paraffin embedded cementoblastoma specimens, when immunostained with anti-CAP mAb, showed strong immunostaining of the cells surrounding the regular and irregularly-shaped calcified masses of the tumor. Putative cementocytes also stained positively. Immunostaining with a polyclonal antibody against osteopontin strongly stained the osteoblastic cells (89.0 +/- 3.6). Cementoblastoma cells showed weaker staining (54.2 +/- 2.4). The results suggest that cementoblastoma cells could be a major source of specific cementum proteins. These cells could provide the opportunity to elucidate the regulation of the cementogenesis process.