Using hDPCs Culture Environments Affect Regenerative Potential of Conditioned Media

Method: DPCs were isolated from healthy human adult third molars that were extracted for clinical purposes. Cells were expanded and cultured in normal culture condition at 37℃ in 5% CO₂/95% air using DMEM supplemented with 10% FCS and 1% antibiotics. At 80% confluency, the media were replaced with serum-free DMEM. Then, DPCs were divided into three groups grown in normoxic, hypoxic and intermittent stretch stress conditions. After 48 hours incubation, CM were collected from each group and applied on several assays. Endothelial cells (human umbilical vein endothelial cells; Lonza), osteoblasts (human transfected fetal osteoblasts; ATCC) and PC12 (neuronal cell derived from rat pheochromocytoma; ATCC) were exposed to the various CM, cultured and incubated in the normal culture condition. At harvest, q-PCR, ELISA, ALP activity, Alizarin red staining and tubeformation, wound healing, apoptosis and neurite outgrowth assays were used to evaluate the effect of the CM on the angiogenic, osteogenic and neurogenic potential of the various cells investigated in the study.

Result: Hypoxic culture condition increased angiogenesis-related gene expression and secretion of angiogenic factors. The angiogenic assays demonstrated that, CM from the hypoxic condition promoted the angiogenic activity. On the other hand, CM from the intermittent stretch group revealed high osteogenic activity and neuroprotective capacity.　

Conclusion: These results suggest that culture conditions significantly influence the regenerative potential of condtioned media aimed for tissue engineering.