http://informahealthcare.com/plt ISSN: 0953-7104 (print), 1369-1635 (electronic) Platelets, Early Online: 1–2 ! 2014 Informa UK Ltd. DOI: 10.3109/09537104.2013.879112

LETTER TO THE EDITOR

Platelet-rich plasma scaffolds for tissue engineering: More than just growth factors in three dimensions Eduardo Anitua, Roberto Prado, Sabino Padilla, & Gorka Orive

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BTI Biotechnology Institute IMASD, Vitoria, Spain

To the editor,

Keywords

We have read with interest the article by Moroz and colleagues [1] regarding the importance of fibronectin in three-dimensional (3D) scaffolds based on platelet-rich plasma (PRP) technology. We agree with the authors that it is essential to properly characterize all biomolecules which are present in this type of bioactive scaffold and which are widely used in tissue engineering. The authors note that one of the challenges of tissue engineering in the field of orthopedics is to produce and be able to mimic cartilage with similar characteristics to the real one, and the use of PRP as a scaffold, either alone or in combination with other biomaterials, is postulated as a valuable option [2]. We would like to offer some additional perspectives on this letter. The importance of plasma molecules present in PRPs has already been described [3], and in this light the biological effects of PRPs cannot be limited to the platelet-derived molecules. It is also complemented by plasma proteins. In particular, fibronectin is a plasma protein that plays a relevant role in hemostasis, wound healing and tissue regeneration [4]. It is also present in the alpha granules of platelets [5], although in proportion, platelet contribution is minimal, so that the enrichment of platelets in PRPs does not substantially increase its concentration [6]. Recently, we carried out a high-throughput proteomic characterization of the fibrin scaffold obtained from plasma rich in growth factors (PRGF), one type of autologous and non-leukocyte containing PRP [7]. Results showed that fibronectin was not just in close contact with fibrin, it was retained in the threedimensional fibrin scaffold. It has been previously reported that 35–40% of fibronectin is retained in the blood clot, due to its binding to fibrin during fibrinogen polymerization [8]. Moreover, it was reported that freezing a supernatant of the degranulated platelets obtained with PRGF can affect the concentration of fibronectin, reducing it by 30% [9]. However, this reduction did not decrease the proliferative and migratory behavior of primary human keratocytes in 2D [9]. Studies should be conducted to observe the effect of this reduction in 3D stem cell cultures. On the other hand, the scaffold described by the authors [10] is based on platelet lysate that freezes the plasma. More studies should be performed to determine whether there is a decrease in the concentration of fibronectin and to describe the optimal formulation in order to prepare the scaffolds. In contrast to platelet lysates, PRGF and other PRP formulations need no prior freezing to form the scaffold and release the platelet content,

Platelet rich plasma, tissue engineering History Received 29 November 2013 Revised 21 December 2013 Accepted 23 December 2013 Published online 10 February 2014

so they are an interesting alternative. PRGF scaffold and supernatant are created by stimulation with an agonist (CaCl2), to prime and degranulate all the platelets and create a 3D scaffold [11]. It would be interesting to further explore whether there is a decrease in fibronectin concentration or in any other molecule using a platelet lysate with respect to PRGF/PRP releasates. The latter could be due to the damage exerted by the freeze-thaw cycles, or because there is an incomplete release of platelet molecules. With the choice to use platelet lysate, there is also variability in blood extraction protocols. In the Moroz et al. study [10], the platelet lysate was prepared by ‘‘fast freezing in liquid nitrogen ( 196  C), then subjected to fast thawing at 37  C to obtain the platelet lysate. The final solution was cryopreserved in liquid nitrogen until MSCs encapsulation’’. There is great variability in the way to obtain the platelet lysate, in particular with respect to freezing and thawing temperatures, or number of cycles of freezing [12–14]. Last but not least, the characteristics of the scaffold can be affected by the selected protocol, since certain proteins involved in both the stabilization and retraction of the fibrin scaffold can be present in a greater or lesser concentration [15]. In particular, the concentration of fibronectin can modulate the three-dimensional structure of the fibrin matrix by adjusting the thickness and density of the fibrin fibers [16]. The implications of these variations in 3D structures in the viability and migration of stem cells must be explored. Previous data suggest that the proliferation of mesenchymal stem cells can be affected by the 3D structure of the fibrin [17]. We encourage the authors to explore these issues, especially concerning the role of fibronectin in chondrogenic differentiation [18], in order to meet the challenge of obtaining cartilage in vitro.

Declaration of interest Correspondence: Gorka Orive, BTI Biotechnology Institute, Vitoria, Spain. E-mail: [email protected]

The authors declare no conflicts of interests. The authors alone are responsable for the content and writing of this article.

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References 1. Moroz A, Felisbino SL, Deffune E. Platelet and plasma bioactive scaffolds for stem cell differentiation: What are we missing? Platelets 2013;[Epub ahead of print]. doi:10.3109/09537104. 2013.836748. 2. Anitua E, Prado R, Orive G. Endogenous morphogens and fibrin bioscaffolds for stem cell therapeutics. Trends Biotechnol 2013;31: 364–374. 3. Anitua E, Sanchez M, Prado R, Orive G. The P makes the difference in plasma rich in growth factors (PRGF) technology. Platelets 2011; 22:473–474. 4. Pankov R, Yamada KM. Fibronectin at a glance. J Cell Sci 2002; 115:3861–3863. 5. Schick PK, Wojensk CM, Bennett V, Denisova L. Fibronectin isoforms in megakaryocytes. Stem Cells 1996;14:212–219. 6. Mosher DF. Plasma fibronectin concentration: A risk factor for arterial thrombosis? Arterioscler Thromb Vasc Biol 2006;26: 1193–1195. 7. Anitua E, Prado R, Azkargorta M, Rodriguez-Suarez E, Iloro I, Casado-Vela J, Elortza F, Orive G. High-throughput proteomic characterization of plasma rich in growth factors (PRGF-Endoret)derived fibrin clot interactome. J Tissue Eng Regen Med 2013;[Epub ahead of print]. doi:10.1002/term.1721. 8. Mccafferty MH, Lepow M, Saba TM, Cho E, Meuwissen H, White J, Zuckerbrod SF. Normal fibronectin levels as a function of age in the pediatric population. Pediatr Res 1983;17:482–485. 9. Anitua E, Muruzabal F, Pino A, Merayo-Lloves J, Orive G. Biological stability of plasma rich in growth factors eye drops after storage of 3 months. Cornea 2013;32:1380–1386. 10. Moroz A, Bittencourt RA, Almeida RP, Felisbino SL, Deffune E. Platelet lysate 3D scaffold supports mesenchymal stem cell

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chondrogenesis: An improved approach in cartilage tissue engineering. Platelets 2013;24:219–225. Anitua E, Prado R, Orive G. Safety and efficient ex vivo expansion of stem cells using platelet-rich plasma technology. Ther Deliv 2013;4:1163–1177. Schallmoser K, Bartmann C, Rohde E, Reinisch A, Kashofer K, Stadelmeyer E, Drexler C, Lanzer G, Linkesch W, Strunk D. Human platelet lysate can replace fetal bovine serum for clinicalscale expansion of functional mesenchymal stromal cells. Transfusion 2007;47:1436–1446. Walenda G, Hemeda H, Schneider RK, Merkel R, Hoffmann B, Wagner W. Human platelet lysate gel provides a novel three dimensional-matrix for enhanced culture expansion of mesenchymal stromal cells. Tissue Eng Part C Methods 2012;18:924–934. Pawitan JA. Platelet rich plasma in xeno-free stem cell culture: The impact of platelet count and processing method. Curr Stem Cell Res Ther 2012;7:329–335. Anitua E, Sanchez M, Prado R, Orive G. The type of platelet-rich plasma may influence the safety of the approach. Knee Surg Sports Traumatol Arthrosc 2012;[Epub ahead of print]. doi:10.1007/ s00167-012-2043-1. Ramanathan A, Karuri N. Fibronectin alters the rate of formation and structure of the fibrin matrix. Biochem Biophys Res Commun 2013;[Epub ahead of print]. doi: 10.1016/j.bbrc.2013.11.090. Ho W, Tawil B, Dunn JC, Wu BM. The behavior of human mesenchymal stem cells in 3D fibrin clots: Dependence on fibrinogen concentration and clot structure. Tissue Eng 2006;12: 1587–1595. Singh P, Schwarzbauer JE. Fibronectin and stem cell differentiation – Lessons from chondrogenesis. J Cell Sci 2012; 125:3703–3712.

Platelet-rich plasma scaffolds for tissue engineering: more than just growth factors in three dimensions.

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