Oxidative Stress-Induced Senescence Responses In Dental Pulp Progenitor Cells

Dental pulp may provide a potential source of progenitor cells for tissue engineering purposes. However, these require considerable in vitro expansion before use, leading to loss of proliferative lifespan and cellular senescence, potentially accompanied by increased oxidative stress, altered cellular responses and differentiation. Objectives: To investigate the effects of oxidative stress on dental pulp progenitor cell (DPPC) clones, in terms of population doubling levels (PDLs) and senescence. Methods: DPPCs were isolated from three wisdom teeth, pulps dissected and cell suspensions obtained by collagenase/dispase digestion. DPPCs were isolated by fibronectin adhesion and clonal cell lines produced. DPPCs were seeded at 1x10⁵ and maintained in the absence/presence of sub-lethal doses of H₂O₂ (5, 10, 25, 50, 100, 200μM). PDLs throughout culture were monitored, in addition to the expression of a triplicate of stem cell markers (CD105, CD90, CD73) by RT-PCR, at different PDLs. Cellular senescence was determined by senescence associated-β-gal staining and telomere restriction fragment (TRF) analysis. Results: DPPC PDLs over 200 days in low H₂O₂ concentrations (5-25μM) suggested no significant alterations in proliferation compared to untreated cultures, although proliferation rates decreased in higher H₂O₂ concentrations (50-200μM) over 80 days. Stem cell marker expression was maintained at 30 days in H₂O₂-treated and control cultures. After 80 days, senescence associated-β-gal staining was higher (61%) in DPPCs cultured in 200μM H₂O₂, compared with untreated controls (28%). Senescence was confirmed by TRF analysis. Differences in the rates of H₂O₂-induced senescence between clones, was also observed. Conclusions: DPPCs are sensitive to H₂O₂ at high (50-200μM) concentrations, leading to a reduction in proliferation, although cells continue to express stem cell markers. Initial data also suggests differential DPPC clone responses to oxidative stress-induced senescence, suggesting variable DPPCs populations, in terms of proliferation and regenerative potential. Further studies will investigate cell behaviour under these extreme oxidative stress (50-200μM) conditions.