The periodontal ligament (PDL) has a reservoir of mesenchymal stem cells
The periodontal ligament (PDL) has a reservoir of mesenchymal stem cells (MSCs) and this tissue is easily available following teeth removal procedures. in mice calvaria were observed. When anoxic PDLCs were subcultured into tendo/ligamentogenic medium, expression of (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. and genes in surviving DPCs after short-time (6 to 24 hours) exposure of anoxic conditions (O2<0.1%).13 Short-time treatment with low-oxygen can be easily applied to cells during the culture process for future clinical use. Therefore, we hypothesized that short-time exposure of the proper concentration of low oxygen may enrich the subpopulation of PDL stem/progenitor cells or enhance the plasticity of cultured PDLCs even at later passages in culture. Here, we analyzed stem cell properties and regenerative capabilities of surviving PDLCs post exposure to transient low-oxygen conditions such as either hypoxic (O25%) vonoprazan or anoxic (O2 0.1%) conditions. Materials and Methods Preparation of PDLCs The procedure used to harvest the extracted teeth from humans conformed to tenets of Declaration of Helsinki, and protocol was approved by Ethics Committee of Nagasaki University. All subjects provided written informed consent. Human periodontal ligament tissues were harvested from healthy normal third molars (5 patients, 3 males and 2 females; average 31years old) after tooth extraction. PDL tissue was mechanically removed by a scalpel, and a single-cell suspension was obtained enzymatically by digesting with 2?mg/mL collagenase (Worthington Biochem) for 1 hour in Dulbecco's modified Eagle's medium (DMEM; Cat No. 041-29775, Sigma) containing 10% fetal bovine serum (FBS; Thermo Trace Ltd) and 2 % antibioticCantimycotic solution on a shaker at 37C. Isolated cells were cultured in medium (DMEM containing 10 % FBS and 2% antibioticCantimycotic solution). When cells reached 80%C90 % confluence, they were subcultured until passage 6 (P6) (population doubling level 24) for subsequent experiments. Low-oxygen exposure Low-oxygen conditions for cell culture were achieved by use of an anaerobic jar and AnaeroPack (Mitsubishi Gas Chemical) for hypoxia (5% O2) and anoxia (0.01% O2). For experiments, P5CP6, PDLCs were seeded at concentration of 5104 cells/well into 24-well plates (Thermo Fischer Scientific, Roskilde) and were cultured until 80%C90% confluence. Then, they were exposed to vonoprazan hypoxic/anoxic conditions in low-glucose/nonserum culture medium (DMEM containing 2% antibioticCantimycotic solution) for 24 hours. Regarding this condition, we performed preliminary experiments to determine the optimum vonoprazan concentrations of glucose and exposure time of cells to low-oxygen as described in our previous work.13 Briefly, cell viability was measured at three different concentrations (non, 0?g/L; low, 1?g/L; and high, 4.5?g/L) in normoxic conditions to determine the glucose concentration, and it was also examined in hypoxic/anoxic conditions every 6?h within 48?h to determine the exposure time for low-oxygen. As a result, low glucose concentration was selected for this study, as it was effective for cell viability under serum-free and normoxia conditions when compared with nonglucose or high-glucose concentrations (data not shown). For the length of exposure to low-oxygen tensions, almost Rabbit polyclonal to WWOX all cells died in anoxic conditions after 36 hours, while more than 50 % of cells could survive within 24 hours (data not shown). Therefore, the evaluations of this study were performed mainly at 24 hours as transient low-oxygen exposures, except the gene expression analysis.