gms | German Medical Science

Objectives: Clinical application of platelet-rich plasma (PRP) and stem cells has become more and more important in regenerative medicine during the last decade. However, differences in PRP preparations result in diverse PRP compositions with unpredictable effects on a cellular level. It has been suggested that leukocyte depletion in PRP may result in a product suitable for regeneration of tissue injuries without formation of scar tissue. In the present study, we evaluated the expression of transcription factors in adipose-tissue derived mesenchymal stem cells (ASCs) while exposed to leukocyte-reduced PRP.

Methods: Leukocyte-reduced PRP was obtained after modification of the centrifugation settings of a commercially available PRP preparation kit (Arthrex Double Syringe, Arthrex, Inc, Naples, Florida). ASCs were harvested from 5 patients and expanded in a-MEM supplemented with 20% FCS until confluent. As passage 1, ASCs were cultured with autologous 10% or 20% PRP or 20% FCS for 48 hours. Cell cycle kinetics of ASCs were analyzed using flow cytometric analyses to assess proliferation. 214 proteins were evaluated by reverse-phase protein array (RPPA). Pathway information was obtained at the Kyoto Encyclopedia of Genes and Genomes (KEGG) and consequently 5 key proteins were selected and confirmed by Western Blot (WB). Paired-Samples T-Test with Bonferroni correction was applied to identify differences in the cell cycle analysis, and One-Way ANOVA with Bonferroni Post Hoc Test was applied to identify differences in expression alteration in the different cell culture conditions.

Results and Conclusion: ASCs exposed to 20% PRP demonstrated a trendency towards a higher percentage of S-phase cells when compared to 10% PRP or 20% FCS (both P>0.14). On the contrary, a lower percentage of cells was observed in the G1-phase in the presence of 20% PRP in comparison to 10% PRP and 20% FCS (both P>0.07). Comparable percentages of cells in the G2-phase were observed in all three media (P>0.22).

We studied the RAS, MAPK, PI3K and mTOR signaling pathway as central pathways involving cell growth and proliferation with PDGFR, MEK 1, c-Myc, PTEN and mTOR as key proteins. RPPA revealed significantly increased expression of MEK 1 and c-Myc in the presence of 20% PRP in comparison to 10% PRP or 20% FCS (both P<0.01). On the contrary, a significantly decreased expression of PDGFR, PTEN and mTOR was demonstrated in the presence of 20% PRP in comparison to 10% PRP or 20% FCS (both P<0.05). WB confirmed trend-setting expression only for MEK 1, c-Myc, PDGFR and PTEN, however opposed expression for mTOR.

These findings suggest a dose-dependent proliferation stimulation of ASCs when cultured in in the presence with autologous PRP. We identified proteins involved in the pathway biology of the interaction between PRP and ASCs. Our results assist in safeguarding the combination of leukocyte-reduced PRP and stem cells for regenerative therapies.