Characterization of the cytokine profile of platelet rich plasma (PRP) and PRP-induced cell proliferation and migration: Upregulation of matrix metalloproteinase-1 and -9 in HaCaT cells

Abstract

Background: The underlying rationale of platelet rich plasma (PRP) therapy is that an injection of concentrated PRP at the site of injury may promote tissue repair via cytokine release from platelets. The molecular mechanisms of PRP therapy in the skin wound healing process are not well understood at present, and would benefit from clarification.

Methods: PRP was stimulated with angonists for 5 min, and cytokine profile analysis was performed. To investigate the wound healing activity of PRP, cell proliferation and migration analyses were performed in skin cells. The effects of PRP were analyzed on the expression and activity of matrix metalloproteinase (MMP)-1, -2, -9, and the activation of transcription factors.

Results: Thrombin was found to be a strong stimulator of PRP activation to release growth factors and chemokines. PRP induced cell proliferation and migration in HUVECs, HaCaT cells, and HDFs, as well as MMP-1and MMP-9 expression in HaCaT cells, but PRP did not have a significant effect on the expression or activity of MMPs in HDFs. The transcription factors, including signal transducer and activator of transcription-3 (STAT-3) were found to be phosphorylated following PRP treatment in HaCaT cells.

Conclusion: In this study, we have identified the cytokine profile of activated PRP after agonist stimulation. We have shown that PRP plays an active role in promoting the proliferation and migration of skin cells via the regulation of MMPs, and this may be applicable to the future development of PRP therapeutics to enhance skin wound healing.

Keywords: Cell migration; Cell proliferation; Cytokine profile; Matrix metalloproteinase; Platelet rich plasma (PRP); Wound healing.

Fig. 1

Cytokine profiles released by PRP after agonist stimulation. One mL of resting PRP containing 1.5×10⁹ platelets was stimulated with 5 µM ADP, 20 µg/mL collagen, 1% Ca²⁺, or 20 U/mL thrombin for 5 min. The released growth factors (A), chemokines (B), and TGF-βs (C) were measured using a multiplex human cytokine/chemokine immunoassay system.

Fig. 2

Effects of PRP on cell proliferation. HUVECs (A) and HaCaT cells (B) were starved with serum-free medium for 24 hr, and then replaced with fresh serum-free medium and incubated for 24 hr with 200 µL of PRP (10% v/v), which had previously been stimulated with 5 µM ADP, 20 µg/mL collagen, 1% Ca²⁺, or 20 U/mL thrombin for 5 min. Cells were then harvested and counted by a hemocytometer. Each treatment was performed in triplicate, and data are presented as means±S.D.

Fig. 3

Effects of PRP on the migration of skin cells. Scratch wounds were created in cell monolayers of HaCaT cells (A) and HDFs (B) using a sterile pipette tip. After washing away suspended cells, cells were replaced with fresh serum-free medium and 200 µL of PRP was added (10% v/v) for 24 hr. The extent of wound closure was presented as the percentage by which the original scratch width had decreased at each measured time point. Experiments were performed in duplicate, and data are presented as means±S.D.

Fig. 4

Effects of PRP on the activity of MMP-1, 2, and 9 in skin cells. HaCaT cells (A) and HDFs (B) were starved with serum-free medium for 24 hr, then replaced with fresh serum-free medium and incubated with 200 µL of PRP (10:1, v/v) for 48 hr. The medium was concentrated, MMP-2 and MMP-9 activities were analyzed by gelatin zymography, and MMP-1 expression was analyzed by Western blotting.

Fig. 5

Effects of PRP on the activation of STAT-3, JNK, and NF-κB in HaCaT cells. HaCaT cells were starved with serum-free medium for 48 hr, and fed with by medium replacement with fresh serum-free medium. Cells were then treated with 200 µL PRP (10% v/v) for 0, 5, 15, 30, 60, or 180 min. Cell lysates were analyzed by Western blotting with anti-phospho-STAT-3, phospho-JNK, and phospho-NF-κB antibodies.

Fig. 6

A schematic diagram of the mechanisms by which PRP promotes wound healing at sites of injury.