Influence of Hypoxic Condition on Cytotoxicity, Cellular Migration, and Osteogenic Differentiation Potential of Aged Periodontal Ligament Cells

Abstract

Objective: This study aimed to investigate and compare the influence of hypoxic conditions on cytotoxicity, cellular migration, and osteogenic differentiation of aged periodontal ligament (PDL) cells.

Materials and methods: Isolated human PDL cells from aged and young subjects were cultured under hypoxic conditions, which were treated with hydrogen peroxide (H₂O₂) (0, 25, 50, 100, 200, and 500 µM). To assess cytotoxicity, lactate dehydrogenase release was determined by the optical density at 490 nm, and the percentage of cell death was calculated. An in vitro wound healing assay was performed over 24 to 48 hours for cellular migration determination. Osteogenic differentiation was determined by alizarin red staining and osteogenic gene expression, including the expression of runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), and osteopontin (OPN).

Results: There was a significant difference in the percentage of cell death with high hypoxic condition (200 and 500 µM) compared to low hypoxic conditions on both day 1 and 2. The highest cellular migration was depicted at 50 µM in both young and aged groups of the in vitro wound healing assay. Osteogenic gene expression of RUNX2 in the aged group was increased at 25 and 50 µM hypoxic condition at day 7, but the expression was gradually decreased after 14 days. On the contrary, the expression of ALP and OPN in the aged group was increased at day 14. Only OPN had been found to be statistically significantly different when compared with gene expression at day 7 and 14 (p < 0.05). The results showed no statistically significant differences when compared with the young and aged groups in all genes and all concentrations.

Conclusion: The concentration of low hypoxic condition (25-50 µM) was proposed to promote cell viability, cellular migration, and osteogenic differentiation in aged PDL cells. We suggested that the potential of aged PDL cells for use in cell therapy for periodontal regeneration might possibly be similar to that of young PDL cells.

Fig. 1

Senescent assay. ( A ) Image from phase contrast microscope of control (periodontal ligament [PDL] cell line), ( B ) young group, and ( C ) aged group. ( A – C ) 4× magnification and scale bar = 200 µm. ( D ) Aged group with 10× magnification. Scale bar = 100 µm. ( E ) Percentage of senescent cells in passage 3. ( F ) Cellular proliferation of control, young, and aged groups ( n  = 10).

Fig. 2

Comparing each concentration of cytotoxic assay. ( A ) Chart showed percentage of cytotoxicity on day 1. ( B ) Chart showed percentage of cell death on day 2. The differences in each group were significant as shown by star (*). Concentration of 200 and 500 µM were significantly different from other concentrations ( p  < 0.05) for both of day 1 and day 2 ( n  = 3).

Fig. 3

For cellular migration in each concentration (0, 25, 50, 100, 200, and 500 µM) and day: ( A ) day 1 and ( B ) day 2. Control had a significant difference compared to the aged and young groups at all concentrations on day 1 and 2 ( p  < 0.05). The differences were significant as shown by star (*). Pictures depicted in vitro wound healing staining with Giemsa ( C ) control, ( D ) young, and ( E ) aged group (scale bar = 200 µm, n  = 10).

Fig. 4

Quantification of alizarin red staining (ARS). ( A ) At 7 days, the concentration of ARS (mM) for 50 µM concentration of both aged and young groups were significantly different when compared to 500 µM ( p  = 0.016 and 0.016, respectively). ( B ) At 14 days, the concentration of ARS for all concentrations were not significant ( p ≥ 0.05, scale bar = 200 µm, n  = 3).

Fig. 5

Gene expression of ( A ) hypoxia-inducible factor-1 alpha (HIF-1α) on day 7, ( B ) HIF-1α on day 14, ( C ) runt-related transcription factor 2 (RUNX2) on day 7, and ( D ) RUNX2 on day 14. The expressions of young and aged group were not different for all concentration ( p ≥ 0.05, n  = 3). ( E ) Alkaline phosphatase (ALP) on day 7 and ( F ) ALP on day 14. The expression of ALP at concentration 25 and 0 µM of the young group was statistically significantly different ( p  = 0.037, n  = 3). ( G ) Osteopontin (OPN) on day 7 and ( H ) OPN on day 14. The OPN gene expression between 7 and 14 days was statistically significantly different ( p  < 0.05, n  = 3).