Enhanced skin wound healing by a sustained release of growth factors contained in platelet-rich plasma

Abstract

Platelet-rich plasma (PRP) contains growth factors that promote tissue regeneration. Previously, we showed that heparin-conjugated fibrin (HCF) exerts the sustained release of growth factors with affinity for heparin. Here, we hypothesize that treatment of skin wound with a mixture of PRP and HCF exerts sustained release of several growth factors contained in PRP and promotes skin wound healing. The release of fibroblast growth factor 2, platelet-derived growth factor-BB, and vascular endothelial growth factor contained in PRP from HCF was sustained for a longer period than those from PRP, calcium-activated PRP (C-PRP), or a mixture of fibrin and PRP (F-PRP). Treatment of full-thickness skin wounds in mice with HCF-PRP resulted in much faster wound closure as well as dermal and epidermal regeneration at day 12 compared to treatment with either C-PRP or F-PRP. Enhanced skin regeneration observed in HCF-PRP group may have been at least partially due to enhanced angiogenesis in the wound beds. Therefore, this method could be useful for skin wound treatment.

Figures

Figure 1

Release of various GFs contained in PRP from various delivery systems. (A) The concentrations of GFs in PRP. Kinetics of release of (B) PDGF-BB, (C) VEGF, and (D) FGF2 from various delivery systems in vitro. Values represent the mean ± standard deviation (n = 3). *P < 0.05 compared to any group. #P < 0.001 compared to any group.

Figure 2

The bioactivity of GFs in PRP released from various delivery systems. Bioactivity of the GFs was evaluated by measuring their ability to stimulate HDF growth in vitro. Values represent the mean ± standard deviation (n = 3). *P < 0.05 compared to any group. #P < 0.01 compared to any group.

Figure 3

Wound closure. (A) Representative photographs of full-thickness skin wounds at various time points after treatment with PRP + various delivery systems. The dotted lines indicate the original wound margin. The dotted circles indicate the knots of stitches. (B) Wound closure rates. *P < 0.05 compared to any other group.

Figure 4

Histological analyses of wound healing. Masson's Trichrome-stained sections of wounds (A) at a low magnification and (B) at a high magnification at 12 days after treatment with PRP + various delivery systems. The dotted squares indicate the mid portion used in (B). Ep, epithelium; De, dermis; h, hair follicle; Gr, granulation tissue; Mu, muscle layer; Sc, subcutaneous tissue; St, scar tissue; He, hypertrophic epithelium. (C) Graphic illustration of wound healing. (D) Wound edge distance (i.e., distance between tips of epithelium), hypertrophic coverage, and inter-SC distance in wounds at 12 days after treatment with PRP + various delivery systems. *P < 0.001 compared to F-PRP or HCF-PRP. **P < 0.05 compared to HCF-PRP.

Figure 5

Epithelial regeneration in full-thickness skin wounds at 12 days after treatment with PRP + various delivery systems. (A) Immunohistochemical staining with anti-involucrin antibodies of treated wound sections. Red and blue (DAPI) indicate differentiated epithelial layer and nucleus, respectively. Scale bars indicate 100 µm. (B) RT-PCR analysis for MMP-9 expression in the wounds at 12 days after treatment with PRP + various delivery systems.

Figure 6

Neovascularization in the wounds at 12 days after treatment with PRP + various delivery systems. Immunohistochemical staining with anti-SM α-actin antibodies. Green and blue (DAPI) indicate the walls of arterioles and nucleus, respectively. Scale bars indicate 100 µm. Arteriole density was quantified. *P < 0.05 compared to any other group.

Figure 7

Neovascularization in the wounds at 12 days after treatment with PRP + various delivery systems. Immunohistochemical staining with anti-vWF antibodies. Green and blue (DAPI) indicate the walls of capillaries and nucleus, respectively. Scale bars indicate 100 µm. The capillary density was quantified. *P < 0.05 compared to any other group.