In vitro formation of mineralized nodules by periodontal ligament cells from the rat

Abstract

The purposes of this study were to determine whether periodontal ligament (PDL) cells are capable of producing mineralized nodules in vitro and to analyze ultrastructural features of the nodules. Rat PDL cells were obtained from coagulum in the socket at 2 days after tooth extraction and cultured at confluence in standard medium containing Dulbecco's Modified Eagle's Medium supplemented with 10% FBS and antibiotics. To test mineralized nodule formation, cells were further cultured for an additional 3 weeks in the standard medium containing (1) ascorbic acid (50 micrograms/ml) and sodium beta-glycerophosphate (10 mM), (2) ascorbic acid, sodium beta-glycerophosphate, and dexamethasone (5 microM), or (3) ascorbic acid alone. Cells were then fixed in 2.5% glutaraldehyde, postfixed in 1% OsO4, and prepared for light and electron microscopy. Three-dimensional nodules containing mineralized matrices were formed only when the cells were cultured in the presence of ascorbic acid and dexamethasone. They were composed of multilayered fibroblasts (up to 13 layers), and highly organized collagen fibrils with 64 nm cross-banding patterns between the cell layers. The fibroblasts in the nodules exhibited an elongated shape with a high degree of cytoplasmic polarity throughout the nodule, and have the morphological features of PDL fibroblasts as seen in vivo. Mineral deposition with needle-like crystals was initiated on collagen fibrils located in intercellular spaces of the upper cell layers and became increasingly heavier towards the bottom half of the nodules. X-ray microanalysis and electron diffraction analysis confirmed that mineral deposition contained calcium and phosphate in the form of immature hydroxyapatite.(ABSTRACT TRUNCATED AT 250 WORDS)

Morinda citrifolia leaves enhance osteogenic differentiation and mineralization of human periodontal ligament cells

“…There was also an absence of nodule formation and matrix mineralization: only aggregated cells with light alizarin red S staining was observed for hPDL cells treated with ascorbic acid and β-GP. These results well agreed with previous studies which suggested that nodule formation of hPDL cells mostly occurred in certain condition such as in the presence of dexamethasone, estradiol, or EMD with TGF-β1 13,29,30) . To confirm the potency of noni leaf extract in inducing osteogenic differentiation and matrix mineralization, a mouse preosteoblast cell line (MC3T3-1) was treated with the optimal concentration of 2.5% of noni leaf extract.…”

“…The ability to grow and manipulate stem cells within the periodontal ligament is of considerable clinical significance, especially for developing novel mechanisms to achieve bone and periodontal tissue regeneration 28) . Previous findings have indicated that hPDL cells possess several osteoblast-like properties, including the ability to form mineralized nodules in vitro 13,29) when cultured in the presence of growth factors such as enamel matrix derivative (EMD) and transforming growth factor-beta1 (TGF-β1) 30) . However, not only is EMD costly, clinical and cell culture studies have yielded controversial and conflicting results 32) .…”

“…As an indication of their ability to promote mineralization, PDL cells produced the alkaline phosphatase (ALP) enzyme in response to stimulation by cytokines and growth factors such as parathyroid hormone, insulin-like growth factor, bone morphogenic proteins, and 1,25-dihydroxyvitamin D 3 [8][9][10][11][12] . Bone-like and/or cementum-like mineralized nodules were formed when cultured in a medium containing ascorbic acid and dexamethasone 13,14) . Currently, diverse osteoconductive and osteoinductive bone graft substitutes -in place of autografts-are commercially available to promote cementum, PDL, and alveolar bone regeneration in periodontal therapy 15,16) .…”

PLAP-1/Asporin, a Novel Negative Regulator of Periodontal Ligament Mineralization

“…Recently it has been revealed that PDL tissue possesses multipotential mesenchymal stem cells that can differentiate into mineralized tissue-forming cells such as osteoblasts and cementoblasts (2,3). In fact, in vitro maintained PDL cells have various osteoblast-like properties, including the capacity to form mineralized nodules, expression of bone-associated markers, and response to bone-inductive factors such as bone morphogenetic protein 2 (BMP-2) (4,5). Interestingly, however, PDL tissue is never ossified in vivo under normal circumstances.…”

Establishment of immortalized clonal cells derived from periodontal ligament cells by induction of the hTERT gene

“…Based on these findings, osteogenic and non-osteogenic cell lines appeared to have been isolated from the original heterogeneous HPL population. Since HPL cells form bone-like mineralized nodules in the presence of ascorbic acid, β-glycerophosphate and dexamethasone in monolayer cultures (29,30), we evaluated the mineralization activity of the original HPL cells and the 2 clones expressing the highest and lowest of ALP activity (A4 and C10, respectively). We also looked for mRNA expression of osteogenic markers ALP, Cbfa1, BMP-2, BMP-4, and OCN, each of which has a unique distribution within the periodontium, and plays important role in cementogenesis and osteogenesis (31,32).…”