OBJECTIVE
To study the age-adjusted regenerative potential of platelet-rich plasma (PRP) in a culture of dermal fibroblast cells with analysis of proliferative, migratory and metabolic activity of cells after their stimulation with PRP from donors of different ages.
MATERIAL AND METHODS
To prepare PRP, we used one of the classic protocols for one-stage centrifugation of whole blood from healthy donors divided into age groups: the first group (n=10) — donors aged 30—40 years, the second group (n=10) — donors aged 60—70 years. We used hTERT-HDFa (d220) cell line and added donor PRP to the experimental wells at concentrations of 10%. Samples without PRP comprised group 0 (K). The same medium without PRP supplemented with 10% FBS was used as a positive control (group 0 (K 10% FBS)). We analyzed metabolic activity of cells (MTT test) and migration activity of fibroblasts in “scratch assay”. We estimated intensity of reactive oxygen species (ROS) release, morphological characteristics of cells and mechanisms of cell death (fluorescence microscopy).
RESULTS
PRP from donors aged 60—70 years reduced metabolic activity of hTERT-HDFa culture on the first day of experiment by 85.5% (p<0.001) compared to the first group. ROS release by fibroblasts after addition of plasma from donors aged 30—40 years was maximum in the first hours and decreased throughout the follow-up period. After addition of plasma from donors aged 60—70 years, ROS release did not show significant changes in the first hours, but significantly increased throughout the follow-up period. Microscopy revealed the highest percentage of viable cells in groups 0 (K 10% FBS) and 1 (donors aged 30—40 years). The highest percentage of necrosis and apoptosis was recorded in group 2 (donors 60—70 years).
CONCLUSION
In the first 24 hours, we revealed pro-oxidant stimulating effect of PRP from young donors with increase in metabolic activity and no cell necrosis. Addition of PRP from elderly donors on the first day was accompanied by lower metabolic activity of culture and higher percentage of cell necrosis and apoptosis. There was no difference in migration activity of cells depending on donor age.
