Downregulation of RANKL and RANKL/osteoprotegerin Ratio in Human Periodontal Ligament Cells During Their Osteogenic Differentiation

Background And Objective: Human periodontal ligament cells (hPDLCs) are considered the promising seed cells in periodontal tissue engineering. Previous studies have discovered the ability of hPDLCs in alveolar bone formation. It remains unclear, however, how the expression of factors associated with osteoclastogenesis in hPDLCs change during their osteogenic differentiation.

Objective: The present study aimed to observe the regulation of receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG) in hPDLCs during their osteogenic differentiation.

Material And Methods: hPDLCs were treated with (M group) or without (C group) the osteogenic induction medium. Alkaline phosphatase activity was displayed with the Gomori calcium phosphate method. Mineralized nodules were detected with von Kossa staining. Expression levels of RANKL and OPG in hPDLCs were analyzed with real-time reverse transcription-polymerase chain reaction and western blot. Tartrate-resistant acid phosphatase (TRAP) staining was used to display the TRAP activity in Raw264.7 cells co-cultured with hPDLCs in the M group and the C group.

Results: We found that alkaline phosphatase staining was shown to be remarkably higher in the M group than that in the C group during the 21 d interval. Mineralized nodules could be seen in the M group but not in the C group. The expression levels of RANKL mRNA significantly decreased in the M group by 1.69-fold (p = 0.096) at day 7, by 2.04-fold (p = 0.000) at day 14 and by 1.84-fold (p = 0.023) at day 21, compared with the corresponding levels of RANKL in the C group. Similarly, the levels of RANKL protein decreased in the M group by 1.82-fold (p = 0.062) at day 7, by 5.64-fold (p = 0.000) at day 14 and by 4.84-fold (p = 0.000) at day 21. The mRNA and protein expression levels of OPG tended to increase in the M group. As a result, the RANKL/OPG mRNA and protein ratios were significantly downregulated by osteogenic induction. In addition, the number of TRAP staining-positive multinuclear cells in the M group was significantly less than in the C group (p = 0.018).

Conclusion: hPDLCs may help inhibit the resorption of alveolar bone during their osteogenic differentiation by reducing the RANKL expression and the RANKL/OPG ratio.