Keywords
Fibrin
Blood platelets
Platelet-rich fibrin
How to Cite
Abstract
Aim: Venous blood derivatives (VBDs) have been suggested as substitutes for Fetal Bovine Serum (FBS) to improve the clinical transition of cell-based therapies. The literature is not clear about which is the best VBDs substitute. The present study aimed to evaluate the influence of VBDs on cell viability and describe a new method to seed these cells in a 3D Platelet-Rich Fibrin (PRF). Methods: Blood was processed to obtain Platelet-Poor Plasma from PRF (P-PRF), Human Serum (HS), Platelet-Poor Plasma from PRP (P-PRP), activated-PRP (a-PRP), and Platelet lysate (PL). Cells were supplemented with each VBD at 10% and FBS at 10% was the control. Cell viability (fibroblast 3T3/NIH) test was evaluated with MTT assay in two ways: i) cell-seeded and expanded with VBD; ii) cell-seed with FBS and expanded with VBD. To seed the Fibrin construct, cells were suspended in PBS and dropped into the blood sample before performing Choukroun’s protocol for PRF. Constructs were cultured for 7 days in VBD supplements and FBS. Histological and Immunohistochemical analysis with vimentin was performed. Cell viability was analyzed by one-way ANOVA. Results: VBD’s production time was very heterogeneous. Cells expanded in HS and a-PRP has grown faster. VBD-supplemented culture media provided cell culture highly sensible to trypsin/EDTA 0.25%. Cells seeded and expanded with VBD presented viability comparable to FBS in HS, a-PRP, and P-PRP (p>0.05) and lower in P-PRF and PL groups (p<0.05). The viability of cell seed with FBS and expanded with VBD was similar between P-PRF, a-PRP, PL, and FBS (p>0.05) and lower in HS and P-PRP (p<0.005). PRF-seeded cells showed a positive expression of vimentin and were able to maintain all cells supplemented with VBD. Conclusion: VBD supplements were able to maintain fibroblast cells in 2D and 3D cultures. The new method of the fibrin-cell construct was efficient to insert the cells into the fibrin network.
References
Conde MC, Chisini LA, Demarco FF, Nör JE, Casagrande L, Tarquinio SB. Stem cell-based pulp tissue engineering: variables enrolled in translation from the bench to the bedside, a systematic review of literature. Int Endod J. 2016 Jun;49(6):543-50. doi: 10.1111/iej.12489.
Chisini LA, Conde MC, Alcázar JC, Silva AF, Nör JE, Tarquinio SB, et al. Immunohistochemical Expression of TGF-β1 and Osteonectin in engineered and Ca(OH)2-repaired human pulp tissues. Braz Oral Res. 2016 Oct 10;30(1):e93. doi: 10.1590/1807-3107BOR-2016.vol30.0093.
Demarco G, Kirschnick L, Watson L, Conde M, Demarco F, Chisini L. What is the clinical applicability of regenerative therapies in dentistry? Rev Gauch Odontol. 2017;65(4):359-67. doi: 10.1590/1981-863720170002000113112
Conde MC, Chisini LA, Grazioli G, Francia A, Carvalho RV, Alcázar JC, et al. Does cryopreservation affect the biological properties of stem cells from dental tissues? a systematic review. Braz Dent J. 2016 Oct-Dec;27(6):633-40. doi: 10.1590/0103-6440201600980.
Jonsdottir-Buch SM, Gunnarsdottir K, Sigurjonsson OE. Human embryonic-derived mesenchymal progenitor cells (hES-MP Cells) are fully supported in culture with human platelet lysates. Bioengineering (Basel). 2020 Jul;7(3):75. doi: 10.3390/bioengineering7030075.
Aussel C, Busson E, Vantomme H, Peltzer J, Martinaud C. Quality assessment of a serum and xenofree medium for the expansion of human GMP-grade mesenchymal stromal cells. PeerJ. 2022 May;10:e13391. doi: 10.7717/peerj.13391.
Burnouf T, Strunk D, Koh MB, Schallmoser K. Human platelet lysate: replacing fetal bovine serum as a gold standard for human cell propagation? Biomaterials. 2016 Jan;76:371-87. doi: 10.1016/j.biomaterials.2015.10.065.
Bieback K. Platelet lysate as replacement for fetal bovine serum in mesenchymal stromal cell cultures. Transfus Med Hemother. 2013 Oct;40(5):326-35. doi: 10.1159/000354061.
Hemeda H, Giebel B, Wagner W. Evaluation of human platelet lysate versus fetal bovine serum for culture of mesenchymal stromal cells. Cytotherapy. 2014 Feb;16(2):170-80. doi: 10.1016/j.jcyt.2013.11.004.
Chisini LA, Conde MCM, Grazioli G, Martin ASS, Carvalho RV, Nör JE, Demarco FF. Venous blood derivatives as FBS-substitutes for mesenchymal stem cells: a systematic scoping review. Braz Dent J. 2017 Nov-Dec;28(6):657-68. doi: 10.1590/0103-6440201701646.
Mannello F, Tonti GA. Concise review: no breakthroughs for human mesenchymal and embryonic stem cell culture: conditioned medium, feeder layer, or feeder-free; medium with fetal calf serum, human serum, or enriched plasma; serum-free, serum replacement nonconditioned medium, or ad hoc formula? All that glitters is not gold! Stem Cells. 2007 Jul;25(7):1603-9. doi: 10.1634/stemcells.2007-0127.
Dolley-Sonneville PJ, Romeo LE, Melkoumian ZK. Synthetic surface for expansion of human mesenchymal stem cells in xeno-free, chemically defined culture conditions. PLoS One. 2013 Aug;8(8):e70263. doi: 10.1371/journal.pone.0070263.
Haque N, Kasim NH, Rahman MT. Optimization of pre-transplantation conditions to enhance the efficacy of mesenchymal stem cells. Int J Biol Sci. 2015 Feb;11(3):324-34. doi: 10.7150/ijbs.10567.
Barro L, Nebie O, Chen MS, Wu YW, Koh MB, Knutson F et al. Nanofiltration of growth media supplemented with human platelet lysates for pathogen-safe xeno-free expansion of mesenchymal stromal cells. Cytotherapy. 2020 Aug;22(8):458-72. doi: 10.1016/j.jcyt.2020.04.099.
Liau LL, Hassan MNFB, Tang YL, Ng MH, Law JX. Feasibility of human platelet lysate as an alternative to foetal bovine serum for in vitro expansion of chondrocytes. Int J Mol Sci. 2021 Jan 28;22(3):1269. doi: 10.3390/ijms22031269.
Pasztorek M, Rossmanith E, Mayr C, Hauser F, Jacak J, Ebner A, et al. Influence of platelet lysate on 2D and 3D amniotic mesenchymal stem cell cultures. Front Bioeng Biotchnol. 2019 Nov 15;7:338. doi: 10.3389/fbioe.2019.00338.
Mujawar S, Iyengar K, Nadkarni S, Mulherkar R. Expansion and characterization of cells from surgically removed intervertebral disc fragments in xenogen-free medium. J Biosci. 2020;45:108.
Koellensperger E, Bollinger N, Dexheimer V, Gramley F, Germann G, Leimer U. Choosing the right type of serum for different applications of human adipose tissue-derived stem cells: influence on proliferation and differentiation abilities. Cytotherapy. 2014 Jun;16(6):789-99. doi: 10.1016/j.jcyt.2014.01.007.
Pham PV, Vu NB, Pham VM, Truong NH, Pham TL, Dang LT, et al. Good manufacturing practice-compliant isolation and culture of human umbilical cord blood-derived mesenchymal stem cells. J Transl Med. 2014 Feb;12:56. doi: 10.1186/1479-5876-12-56.
Harrison P, Cramer EM. Platelet alpha-granules. Blood Rev. 1993 Mar;7(1):52-62. doi: 10.1016/0268-960x(93)90024-x.
Choukroun J, Diss A, Simonpieri A, Girard MO, Schoeffler C, Dohan SL, et al. Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part IV: clinical effects on tissue healing. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006 Mar;101(3):e56-60. doi: 10.1016/j.tripleo.2005.07.011.
Kawase T. Platelet-rich plasma and its derivatives as promising bioactive materials for regenerative medicine: basic principles and concepts underlying recent advances. Odontology. 2015 May;103(2):126-35. doi: 10.1007/s10266-015-0209-2.
Parihar AS, Narang S, Dwivedi S, Narang A, Soni S. Platelet-rich fibrin for root coverage: a plausible approach in periodontal plastic and esthetic surgery. Ann Afr Med. 2021 Jul-Sep;20(3):241-4. doi: 10.4103/aam.aam_31_20.
Starzyńska A, Kaczoruk-Wieremczuk M, Lopez MA, Passarelli PC, Adamska P. The growth factors in advanced platelet-rich fibrin (A-PRF) reduce postoperative complications after mandibular third molar odontectomy. Int J Environ Res Public Health. 2021 Dec;18(24):13343. doi: 10.3390/ijerph182413343.
Conde MCM, Chisini LA, Sarkis-Onofre R, Schuch HS, Nör JE, Demarco FF. A scoping review of root canal revascularization: relevant aspects for clinical success and tissue formation. Int Endod J. 2017 Sep;50(9):860-74. doi: 10.1111/iej.12711.
Angerame D, De Biasi M, Kastrioti I, Franco V, Castaldo A, Maglione M. Application of platelet-rich fibrin in endodontic surgery: a pilot study. G Ital Endod. 2015;29(2):51-7. doi: 10.1016/j.gien.2015.08.003.
Chisini L, Grazioli G, Francia A, San Martin AS, Demarco FF, Conde MC. Revascularization versus apical barrier technique with mineral trioxide aggregate plug: a systematic review. G Ital Endod. 2018;32(1):9-16. doi: 10.1016/j.gien.2018.03.006.
Yoshpe M, Kaufman AY, Lin S, Ashkenazi M. Regenerative endodontics: a promising tool to promote periapical healing and root maturation of necrotic immature permanent molars with apical periodontitis using platelet-rich fibrin (PRF). Eur Arch Paediatr Dent. 2021 Jun;22(3):527-34. doi: 10.1007/s40368-020-00572-4.
Pavani MP, Reddy KRKM, Reddy BH, Biraggari SK, Babu CHC, Chavan V. Evaluation of platelet-rich fibrin and tricalcium phosphate bone graft in bone fill of intrabony defects using cone-beam computed tomography: a randomized clinical trial. J Indian Soc Periodontol. 2021 Mar-Apr;25(2):138-43. doi: 10.4103/jisp.jisp_621_19.
Chisini LA, Conde MCM, Grazioli G, Martin ASS, Carvalho RV, Sartori LRM, Demarco FF. Bone, periodontal and dental pulp regeneration in dentistry: a systematic scoping review. Braz Dent J. 2019 Mar-Apr;30(2):77-95. doi: 10.1590/0103-6440201902053.
Kumar N, Prasad K, Ramanujam L, K R, Dexith J, Chauhan A. Evaluation of treatment outcome after impacted mandibular third molar surgery with the use of autologous platelet-rich fibrin: a randomized controlled clinical study. J Oral Maxillofac Surg. 2015 Jun;73(6):1042-9. doi: 10.1016/j.joms.2014.11.013.
Chisini LA, Karam SA, Noronha TG, Sartori LRM, San Martin AS, Demarco FF, et al. Platelet-poor plasma as a supplement for fibroblasts cultured in platelet-rich fibrin. Acta Stomatol Croat. 2017 Jun;51(2):133-40. doi: 10.15644/asc51/2/6.
Gassling V, Douglas T, Warnke PH, Açil Y, Wiltfang J, Becker ST. Platelet-rich fibrin membranes as scaffolds for periosteal tissue engineering. Clin Oral Implants Res. 2010 May;21(5):543-9. doi: 10.1111/j.1600-0501.2009.01900.x.
Chen Y, Niu Z, Xue Y, Yuan F, Fu Y, Bai N. Improvement in the repair of defects in maxillofacial soft tissue in irradiated minipigs by a mixture of adipose-derived stem cells and platelet-rich fibrin. Br J Oral Maxillofac Surg. 2014 Oct;52(8):740-5. doi: 10.1016/j.bjoms.2014.06.006.
Sun CK, Zhen YY, Leu S, Tsai TH, Chang LT, Sheu JJ, et al. Direct implantation versus platelet-rich fibrin-embedded adipose-derived mesenchymal stem cells in treating rat acute myocardial infarction. Int J Cardiol. 2014 May;173(3):410-23. doi: 10.1016/j.ijcard.2014.03.015.
Mojica-Henshaw MP, Jacobson P, Morris J, Kelley L, Pierce J, Boyer M, et al. Serum-converted platelet lysate can substitute for fetal bovine serum in human mesenchymal stromal cell cultures. Cytotherapy. 2013 Dec;15(12):1458-68. doi: 10.1016/j.jcyt.2013.06.014.
Choukroun J AF, Schoeffler C, Vervelle A. Une opportunité en paro-implantologie: le PRF. Implantodontie. 2001 Jan:42:55-62.
Dohan Ehrenfest DM, Bielecki T, Jimbo R, Barbé G, Del Corso M, Inchingolo F, et al. Do the fibrin architecture and leukocyte content influence the growth factor release of platelet concentrates? An evidence-based answer comparing a pure platelet-rich plasma (P-PRP) gel and a leukocyte- and platelet-rich fibrin (L-PRF). Curr Pharm Biotechnol. 2012 Jun;13(7):1145-52. doi: 10.2174/138920112800624382.
Karam S, San Martin A, Mazzetti T, Conde M, Chisini L, Demarco F. [Cryogenic treatment to increase the amount of macropores in plasma rich in fibrin used as scaffold in tissue engineering]. ROBRAC. 2017;26(77):14-9. Portuguese.
Bieback K, Ha VA, Hecker A, Grassl M, Kinzebach S, Solz H, et al. Altered gene expression in human adipose stem cells cultured with fetal bovine serum compared to human supplements. Tissue Eng Part A. 2010 Nov;16(11):3467-84. doi: 10.1089/ten.TEA.2009.0727.
Kocaoemer A, Kern S, Klüter H, Bieback K. Human AB serum and thrombin-activated platelet-rich plasma are suitable alternatives to fetal calf serum for the expansion of mesenchymal stem cells from adipose tissue. Stem Cells. 2007 May;25(5):1270-8. doi: 10.1634/stemcells.2006-0627.
Jung JP, Bache-Wiig MK, Provenzano PP, Ogle BM. Heterogeneous Differentiation of Human Mesenchymal Stem Cells in 3D Extracellular Matrix Composites. Biores Open Access. 2016 Jan;5(1):37-48. doi: 10.1089/biores.2015.0044.
This work is licensed under a Creative Commons Attribution 4.0 International License.
Copyright (c) 2024 Luiz Alexandre Chisini, Marcus Cristian Muniz Conde, Sarah Arangurem Karam, Rodrigo Varella de Carvalho, Sandra Beatriz Chaves Tarquinio, Flávio Fernando Demarco
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