Abstract
The periodontal ligament (PDL) has a reservoir of mesenchymal stem cells (MSCs) and this tissue is easily available following teeth removal procedures. However, PDL-derived cells (PDLCs) availability for tissue engineering is limited because they are heterogeneous cells at various differentiation and lineage commitments. Therefore, efficient culture conditions to increase MSCs number are needed to use PDLCs in tissue engineering. Recent reports indicate that low-oxygen conditions amplified stem/progenitor cell numbers and inhibited cell differentiation. Our aim was to establish which low-oxygen culture conditions favored bone or tendon/ligament regeneration in cultured PDLCs. Human PDLCs were cultured and exposed to either hypoxic (O2≤5%) or anoxic (O2<0.1%) oxygen conditions in low-glucose/serum-free media for 24 hours. After 24 h, as expected, cell survival was significantly less in PDLCs exposed to anoxic conditions as compared with cells under normal or hypoxic conditions. PDLCs exposed to hypoxic conditions had the highest percentages for MSC markers (CD105, CD166, Stro-1). For both hypoxic and anoxic conditions, stem cell marker genes (oct4, sox2, p75) were upregulated after 6 h. At 24 h, these stem cell markers were maintained in PDLCs under hypoxic condition. Interestingly under anoxic conditions, expression of scleraxis gene (a key transcription factor for tendo/ligamentogenesis) was upregulated markedly. When hypoxic PDLCs were subcultured into osteogenic medium, in vitro calcification and prominent in vivo bone formation in mice calvaria were observed. When anoxic PDLCs were subcultured into tendo/ligamentogenic medium, expression of aggrecan (a mature tenogenic gene) increased remarkably. No obvious differences were detectable on chondrogenic and adipogenic inducibilities. We propose that transient exposure to low-oxygen during the culture enhanced MSC population in PDL. In addition, different low-oxygen concentrations favored osteogenic or tendo/ligamentogenic inducibilities of cultured PDLCs.