Comparative effect of platelet-rich plasma, platelet-poor plasma, and fetal bovine serum on the proliferative response of periodontal ligament cell subpopulations
Cell-based therapies involve the need to expand cell cultures ex vivo for their subsequent implantation in an autologous manner. An important limitation regarding this technology is the use of fetal bovine serum (FBS) that has critical safety limitations. Platelet-derived fractions represent an autologous source of growth factors that may be used for the expansion of these cell cultures. Periodontal ligament (PDL) cells comprise a heterogeneous cell population that may not necessarily respond in a uniform manner to proliferative stimuli. The aim of this study was to evaluate the ability of two platelet-derived fractions, platelet-rich plasma (PRP) and platelet-poor plasma (PPP) and FBS on the proliferative response of different subpopulations of PDL cell cultures.
Materials and methods
PDL cells were characterized and then exposed to PRP, PPP, or FBS during 2, 5, or 14 days to analyze cell proliferation and clonogenic capability. Cell proliferation was evaluated through immunofluorescence for Ki67 and by tracing carboxyfluorescein diacetate succinimidyl ester (CFSE) dye in combination with mesenchymal stem cell markers using flow cytometry.
Both PRP and PPP stimulated PDL cell proliferation and their clonogenic ability. We found a significant increase of CD73- and CD90-positive cells after PRP or PPP treatment, compared to FBS. Otherwise, no differences were found regarding the response of CD146-or CD105-positive cells when stimulated with PRP, PPP, or FBS.
PRP and PPP can stimulate the proliferation and clonogenicity of PDL cell populations including cells positive for CD90 and CD73 markers.
These findings may have implications for future therapies aiming to stimulate periodontal regeneration using autologous growth factors.
KeywordsPlatelet-rich plasma Periodontal ligament Mesenchymal stem cells Serum-free culture media
This work was supported by the projects FONDECYT 11121294, PUENTE P1704/2017 (CM), and FONDECYT 1130618 (PS) Millennium Institute on Immunology and Immunotherapy (P09/016-F, to AK). The flow cytometry experiments were performed at the Flow Cytometry Core Facility, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile. We acknowledge the contribution of tissue and blood donors.
The selected hybridoma monoclonal antibodies were PD31, developed by Elizabeth A. Wayner and Gregory Vercellotti and P8B1 by Elizabeth A. Wayner/Tucker LeBien, from the Fred Hutchinson Cancer Research Center, Seattle, WA. These antibodies were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA, 52242.
The work was supported by the Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT) from the Chilean Government and Puente research grant by Pontificia Universidad Católica de Chile.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
All procedures performed in studies involving samples from human participants were in accordance with the ethical standards of the Faculty of Medicine Ethical Committee at Pontificia Universidad Católica de Chile and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Informed consent was obtained from all individual participants included in the study.
- 1.Seo BM, Song IS, Um S, Lee J-H (2015) Chapter 22 periodontal ligament stem cells. Stem cell biology and tissue engineering in dental sciences. Academic Press. https://doi.org/10.1016/B978-0-12-397157-9.00024-2 CrossRefGoogle Scholar
- 9.Karnieli O, Friedner OM, Allickson JG, Zhang N, Jung S, Fiorentini D, Abraham E, Eaker SS, Yong TK, Chan A, Griffiths S, Wehn AK, Oh S, Karnieli O (2017) A consensus introduction to serum replacements and serum-free media for cellular therapies. Cytotherapy 19:155–169. https://doi.org/10.1016/j.jcyt.2016.11.011 CrossRefPubMedGoogle Scholar
- 11.Trubiani O, Piattelli A, Gatta V, Marchisio M, Diomede F, D'Aurora M, Merciaro I, Pierdomenico L, Maraldi NM, Zini N (2015) Assessment of an efficient xeno-free culture system of human periodontal ligament stem cells. Tissue Eng Part C Methods 21:52–64. https://doi.org/10.1089/ten.TEC.2014.0024 CrossRefPubMedGoogle Scholar
- 13.Eaker S, Armant M, Brandwein H, Burger S, Campbell A, Carpenito C, Clarke D, Fong T, Karnieli O, Niss K, Van’t Hof W, Wagey R (2013) Concise review: guidance in developing commercializable autologous/patient-specific cell therapy manufacturing. Stem Cells Transl Med 2:871–883. https://doi.org/10.5966/sctm.2013-0050 CrossRefPubMedPubMedCentralGoogle Scholar
- 16.Diez JM, Bauman E, Gajardo R, Jorquera JI (2015) Culture of human mesenchymal stem cells using a candidate pharmaceutical grade xeno-free cell culture supplement derived from industrial human plasma pools. Stem Cell Res Ther 6:28. https://doi.org/10.1186/s13287-015-0016-2 CrossRefPubMedPubMedCentralGoogle Scholar
- 25.Magalon J, Bausset O, Serratrice N, Giraudo L, Aboudou H, Veran J, Magalon G, Dignat-Georges F, Sabatier F (2014) Characterization and comparison of 5 platelet-rich plasma preparations in a single-donor model. Arthroscopy 30:629–638. https://doi.org/10.1016/j.arthro.2014.02.020 CrossRefPubMedGoogle Scholar
- 29.Ochoa-Gonzalez F, Cervantes-Villagrana AR, Fernandez-Ruiz JC, Nava-Ramirez HS, Hernandez-Correa AC, Enciso-Moreno JA, Castaneda-Delgado JE (2016) Metformin induces cell cycle arrest, reduced proliferation, wound healing impairment in vivo and is associated to clinical outcomes in diabetic foot ulcer patients. PLoS One 11:e0150900. https://doi.org/10.1371/journal.pone.0150900 CrossRefPubMedPubMedCentralGoogle Scholar
- 33.Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop D, Horwitz E (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8:315–317. https://doi.org/10.1080/14653240600855905 CrossRefGoogle Scholar
- 37.Zhu W, Tan Y, Qiu Q, Li X, Huang Z, Fu Y, Liang M (2013) Comparison of the properties of human CD146+ and CD146- periodontal ligament cells in response to stimulation with tumour necrosis factor alpha. Arch Oral Biol 58:1791–1803. https://doi.org/10.1016/j.archoralbio.2013.09.012 CrossRefPubMedGoogle Scholar
- 39.Yang ZX, Han ZB, Ji YR, Wang YW, Liang L, Chi Y, Yang SG, Li LN, Luo WF, Li JP, Chen DD, Du WJ, Cao XC, Zhuo GS, Wang T, Han ZC (2013) CD106 identifies a subpopulation of mesenchymal stem cells with unique immunomodulatory properties. PLoS One 8:e59354. https://doi.org/10.1371/journal.pone.0059354 CrossRefPubMedPubMedCentralGoogle Scholar
- 41.Moraes DA, Sibov TT, Pavon LF, Alvim PQ, Bonadio RS, Da Silva JR, Pic-Taylor A, Toledo OA, Marti LC, Azevedo RB, Oliveira DM (2016) A reduction in CD90 (THY-1) expression results in increased differentiation of mesenchymal stromal cells. Stem Cell Res Ther 7:97. https://doi.org/10.1186/s13287-016-0359-3 CrossRefPubMedPubMedCentralGoogle Scholar
- 42.Anderson P, Carrillo-Galvez AB, Garcia-Perez A, Cobo M, Martin F (2013) CD105 (endoglin)-negative murine mesenchymal stromal cells define a new multipotent subpopulation with distinct differentiation and immunomodulatory capacities. PLoS One 8:e76979. https://doi.org/10.1371/journal.pone.0076979 CrossRefPubMedPubMedCentralGoogle Scholar
- 45.Amable PR, Carias RB, Teixeira MV, da Cruz Pacheco I, Correa do Amaral RJ, Granjeiro JM, Borojevic R (2013) Platelet-rich plasma preparation for regenerative medicine: optimization and quantification of cytokines and growth factors. Stem Cell Res Ther 4:67. https://doi.org/10.1186/scrt218 CrossRefPubMedPubMedCentralGoogle Scholar
- 46.Kaigler D, Avila G, Wisner-Lynch L, Nevins ML, Nevins M, Rasperini G, Lynch SE, Giannobile WV (2011) Platelet-derived growth factor applications in periodontal and peri-implant bone regeneration. Expert Opin Biol Ther 11:375–385. https://doi.org/10.1517/14712598.2011.554814 CrossRefPubMedPubMedCentralGoogle Scholar
- 48.Cochran DL, Oh TJ, Mills MP, Clem DS, McClain PK, Schallhorn RA, McGuire MK, Scheyer ET, Giannobile WV, Reddy MS, Abou-Arraj RV, Vassilopoulos PJ, Genco RJ, Geurs NC, Takemura A (2016) A randomized clinical trial evaluating rh-FGF-2/beta-TCP in periodontal defects. J Dent Res 95:523–530. https://doi.org/10.1177/0022034516632497 CrossRefPubMedGoogle Scholar