gms | German Medical Science

German Congress of Orthopaedics and Traumatology (DKOU 2016)

25.10. - 28.10.2016, Berlin

Leukocyte-reduced platelet-rich plasma stimulates the in vitro proliferation of adipose-tissue derived mesenchymal stem cells depending on PDGF signaling in a time dependent manner

Meeting Abstract

  • presenting/speaker Siegmund Lang - Universitätsklinikum Regensburg, Klinik und Poliklinik für Unfallchirurgie, Regensburg, Germany
  • Alexander Hanke - Universitätsklinikum Regensburg, Klinik und Poliklinik für Unfallchirurgie, Regensburg, Germany
  • Marietta Herrmann - AO Reserach Institute, Davos Platz, Switzerland
  • Michael Nerlich - Universitätsklinikum Regensburg, Klinik und Poliklinik für Unfallchirurgie, Regensburg, Germany
  • Peter Angele - Universitätsklinikum Regensburg, Klinik und Poliklinik für Unfallchirurgie, Regensburg, Germany
  • Lukas Prantl - Zentrum für Plastische-, Hand- und , Wiederherstellungschirurgie, Klinik für Unfallchirurgie, Regensburg, Germany
  • Sebastian Gehmert - Unispital Basel, Orthopädie, Basel, Switzerland
  • Markus Loibl - Universitätsklinikum Regensburg, Klinik und Poliklinik für Unfallchirurgie, Regensburg, Germany

Deutscher Kongress für Orthopädie und Unfallchirurgie (DKOU 2016). Berlin, 25.-28.10.2016. Düsseldorf: German Medical Science GMS Publishing House; 2016. DocGR14-62

doi: 10.3205/16dkou428, urn:nbn:de:0183-16dkou4287

Published: October 10, 2016

© 2016 Lang et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 License. See license information at http://creativecommons.org/licenses/by/4.0/.


Outline

Text

Objectives: Platelet-rich plasma (PRP) has been suggested as a xenoprotein-free cell culture medium supplement for mesenchymal stem cells (MSCs). Stem cell-based therapies require robust MSC expansion and targeted differentiation in verified preclinical cell culture systems. However, only limited knowledge exists regarding PRP's growth factors and their effect on MSCs' fate. This study demonstrates dose- and time-dependent effects of leukocyte-reduced PRP on the proliferation and protein expression of adipose-tissue derived mesenchymal stem cells (ASCs) and evaluates the role of PDGF signaling herein.

Methods: Leukocyte-reduced PRP was obtained with a commercially available PRP preparation kit (Arthrex Double Syringe) from 10 male patients. ASCs were harvested from 4 male patients and expanded in α -MEM supplemented with 20% FCS until confluent. ASCs were starved for 24 hours prior to 72 hours incubation in α -MEM with (a) 10% or (b) 20% pooled PRP or (c) 20% FCS. S-phase fraction (SPF) of ASCs was analysed after 6, 12, 24, 48 and 72 hours using flow cytometric analysis to assess proliferation over time. The influence of 10ng/μl recombinant PDGF subtypes AA, AB and BB on ASC proliferation was measured after 48 hours. 3 μM PDGF receptor β inhibitor was added to ASCs for 30 minutes, followed by α -MEM with 20% FCS or 20% PRP for 48 hours. SPF of ASCs with and without receptor inhibition was determined and PDGF receptor β protein (PDGFR β) expression was measured by immunoblotting.

Results and Conclusion: During the first 24 hours no significant difference in SPF of ASCs in presence of 20% FCS, 10% PRP or 20% PRP was observed. SPF of ASCs exposed to 20% PRP peaked after 48 hours and was significantly higher compared to 20% FCS and 10% PRP (p < 0.01). SPF of ASCs supplemented with 10% PRP dropped significantly after 48 hours and after 72 hours compared to previous values (both p < 0.01). ASCs exposed to recombinant PDGF-AB and -BB demonstrated significantly higher SPF in comparison to PDGF-AA and 20% FCS after 48 hours (p < 0.05). PDGFR β blockage prevents PRP-induced SPF increase in ASCs after 48 hours compared to PRP cultured cells without inhibitor (p < 0.01). Conversely, PDGFR β expression was significantly down-regulated in presence of 20% PRP compared to 20% FCS after 48 hours (p < 0.01). The expression of PDGFR β was significantly higher in 20% PRP cultured cells, when receptor inhibitor was added (p < 0.01).

Proliferation and protein expression of ASCs cultured with 20% PRP supplement is depending on PDGF signaling through PDGFR β . Down-regulation of PDGFR β in ASCs stimulated with PRP should be considered for choosing optimal growth factor concentrations in PRP preparations. Our results suggest, that 20% PRP is an efficient and safe medium supplement for ASC expansion over 48 hours.