Platelet lysate promotes in vitro wound scratch closure of human dermal fibroblasts: different roles of cell calcium, P38, ERK and PI3K/AKT

Abstract

There is a growing interest for the clinical use of platelet derivates in wound dressing. Platelet beneficial effect is attributed to the release of growth factors and other bioactive substances, though mechanisms are mostly unknown. We studied wound‐healing processes of human primary fibroblasts, by exposing cells to a platelet lysate (PL) obtained from blood samples. Crystal violet and tetrazolium salt (MTS) assays showed dose–response increase of cell proliferation and metabolism. In scratch wound and transwell assays, a dose of 20% PL induced a significant increase of wound closure rate at 6 and 24 hrs, and had a strong chemotactic effect. BAPTA‐AM, SB203580 and PD98059 caused 100% inhibition of PL effects, whereas wortmannin reduced to about one third the effect of PL on wound healing and abolished the chemotactic response. Confocal imaging showed the induction by PL of serial Ca2+ oscillations in fibroblasts. Data indicate that cell Ca2+ plays a fundamental role in wound healing even without PL, p38 and ERK1/2 are essential for PL effects but are also activated by wounding per se, PI3K is essential for PL effects and its downstream effector Akt is activated only in the presence of PL. In conclusion, PL stimulates fibroblast wound healing through the activation of cell proliferation and motility with different patterns of involvement of different signalling pathways.

Role of ERK1/2 in platelet lysate‐driven endothelial cell repair

“…Such a view is supported by data from the cell migration assay, which showed a chemoattractant activity of PL on HuVEC. These data confirm previous findings about the chemotactic responses to platelet derivatives of retinal glial cells [Castelnovo et al, 2000], rat foetal cells [Soffer et al, 2003], mature osteoblasts [Celotti et al, 2006], keratinocytes [Ranzato et al, 2008] and primary fibroblasts [Ranzato et al, 2009b].…”

“…Hence, we confirmed the key experiments of this study in human microvascular cells, and also included in the study non-human vascular models, in order to obtain a wider panel of data about the potentialities of PL factors in the stimulation of wound healing. The CV assay highlighted a mitogenic effect of PL, thus confirming previous findings about cell proliferation induced by PL on different cell types [Ranzato et al, 2008[Ranzato et al, , 2009b. Interestingly, the proliferative effect of PL was maximum in microvascular HMVEC-c cells, in which the expression of PDGFR and CXCR3 has been characterised.…”

“…It has been also shown that Ca 2þ rises lead to nitric oxide production, which in turn promotes endothelial cell proliferation [Erdogan et al, 2005]. A correlation between Ca 2þ dynamics and wound repair has been also reported in studies on skin cells, where PL has induced noticeable Ca 2þ signals, and BAPTA-AM has abolished the PL effect on wound healing [Ranzato et al, 2008[Ranzato et al, , 2009b. In our experiments, the role of cell Ca 2þ in HuVEC endothelial repair was revealed by the inhibitory effect on PL of the Ca 2þ chelator BAPTA-AM.…”

“…The PL was obtained as described by Ranzato et al [2008Ranzato et al [ , 2009b. Briefly, platelet concentrates were derived by platelet aphaeresis collection from blood samples of single volunteer donors after informed consent.…”

“…This platelet derivative, which is obtained from repeated freezingthawing of platelet-enriched blood samples, has been shown to accelerate the in vitro wound healing of skin cells [Ranzato et al, 2008[Ranzato et al, , 2009b and mouse myoblasts [Ranzato et al, 2009a]. It has been also used as a dressing in clinical practice to improve wound healing [Mazzucco et al, 2004].…”