We have read with interest the article published by Ramos-Torrecillas et al1 titled "Human Fibroblast-Like Cultures in the Presence of Platelet-Rich Plasma as a Single Growth Factor Source: Clinical Implications" in the March 2014 issue of Advances in Skin & Wound Care. In this interesting article, the authors described the behavior of gingival fibroblasts cultured in the presence of platelet-rich plasma (PRP) as cell culture supplement compared with conventional culture conditions supplemented with fetal bovine serum. Cell proliferation, morphology, and antigenic profile (fibronectin, vimentin, and [alpha]-actin) are assayed in short- (2 passes) and long-term (10 passes) cultures.

The assays conducted by the authors present a correct experimental design. The idea of using PRP as a unique supplement for cell culture, including cell isolation and expansion, is certainly appealing. It could be a challenge to try and test a fully autologous strategy by using the same donor source for both PRP and cells. However, there are several issues that we would like to discuss with the authors.

The results showed an increased [alpha]-actin expression in long-term cultures supplemented with PRP. To try to explain this result, we could focus on the type of PRP used as cell culture supplement. An exciting hypothesis leads us to propose that the type of PRP used in this study can influence this outcome. The authors did not characterize the autologous cocktail of proteins and growth factors used in cell cultures, but in view of centrifugation conditions (double spin including buffy coat), it can be assumed that the PRP contains leukocytes and therefore is a leukocyte-enriched PRP. The inclusion of leukocytes in these types of products generates a proinflammatory milieu that may affect fibroblast culture. Recently, we have observed that leukocytes produce a significant increase in the contents of proinflammatory cytokines interleukins 1[beta] and 16 but not in the platelet-derived growth factors release (<1.5-fold).2 Moreover, the availability of vascular endothelial growth factor suffered an important decrease after inclusion of leukocytes within PRP.2

The induction of myofibroblast phenotype in the presence of PRP is controversial in the literature. Several authors have described this induction, but always using l-PRPs.3-6 Instead, our group uses plasma rich in growth factors (PRGF), a platelet-enriched plasma that is characterized by the absence of leukocytes in its formulation.7 We have found that the in vitro treatment with PRGF inhibits and reverts transforming growth factor (TGF-[beta]1)-induced myodifferentiation, both in gingival fibroblasts8 as in keratocytes and conjunctival fibroblasts.9 Moreover, it has been observed that PRGF treatment in an in vivo model of corneal wound healing reduces the presence of myofibroblasts, facilitating wound healing without scarring.10 The differences found regarding myofibroblasts could be due to the different PRP composition in terms of leukocyte content.

We do agree with the authors that the TGF-[beta] is one of the major inducers of myofibroblastic differentiation. In fact, it is the molecule that we use to induce this cellular phenotype. As the authors stated, there is no doubt that TGF-[beta] is present in the PRP, but the PRP is a complex molecular mixture that, in addition to TGF-[beta], contains other molecules that can counteract this myofibroblast-inductive effect. For example, PRP also contains fibroblast growth factor, a molecule that can reverse the myofibroblastic phenotype,11 and hepatocyte growth factor, which can counteract the TGF-[beta]-mediated induction.8

Finally, in the last paragraph of the Discussion section, the authors assert that "A recent study by Anitua et al reported that PRP prevents and inhibits TGF-[beta]1-induced myofibroblast differentiation" and that "this finding appears to call into question the usefulness of PRP in tissue regeneration, because its inhibition of the function of the TGF-[beta]1 physiologically present in tissues would presumably reduce the generation of myofibroblasts and thereby hinder the wound-healing process, given the key role of myofibroblasts in soft tissue regeneration." In this context, we also consider that the myofibroblast plays a pivotal role in wound healing, but its persistence can generate nonfunctional fibrotic tissues.12 For example, in tissue repair, such as skin wound healing, a controlled and transient activation of myofibroblasts is mandatory to attempt to restore the tissue integrity.13 We do not believe that the increase in myofibroblasts in long-term cultures would be positive for wound healing.1 Recently, Caceres et al14 have shed light on the molecular mechanisms whereby a defective wound healing occurs with age. Surprisingly, gingival fibroblast cultures from older donors showed the largest percentage of spontaneous myofibroblastic differentiation than cell cultures of young donors. These data are consistent with the idea that the presence of myofibroblasts should be transient and that in order to achieve optimal wound healing the number of myofibroblasts must be reduced or even disappear at later stages of wound healing.15

We would like to congratulate the authors for this comparative study and encourage them to conduct further research in this area to decipher PRP action mechanisms in order to optimize treatment of patients.

-Eduardo Anitua, MD, PhD; Roberto Prado, BS; and

Gorka Orive, MD, PhD

Foundation Eduardo Anitua, Vitoria, Spain

In response:

Our results are related to investigations by Anitua. In our study, we show the mechanisms that could increase the proliferation and differentiation of fibroblasts tomyofibroblasts by, inter alia, the transforming growth factor A. Wewould like to congratulate the authors for this letter to the editor, and we encourage them to continue research in regenerative therapies with plateletrich plasma.

-Javier Ramos-Torrecillas, PhD;

Elvira de Luna-Bertos, PhD;

Francisco J. Manzano-Moreno, MS;

Olga Garcia-Martinez, PhD; and

Concepcion Ruiz, PhD

University of Granada, Spain

References