Clin Orthop Surg.  2018 Jun;10(2):240-247. 10.4055/cios.2018.10.2.240.

Leukocyte-Poor Platelet-Rich Plasma-Derived Growth Factors Enhance Human Fibroblast Proliferation In Vitro

• Noh KC
• Liu XN
• Zhuan Z
• Yang CJ
• Kim YT
• Lee GW
• Choi KH
• Kim KO

• KMID: 2411751
• DOI: http://doi.org/10.4055/cios.2018.10.2.240

Abstract

BACKGROUND
Leukocyte-poor platelet-rich plasma (LP-PRP) from peripheral blood is currently used as a concentrated source of growth factors to stimulate repair at sites of soft tissue injury. Fibroblasts are primary mediators of wound healing. Thus, we aimed to assess the positive effect of LP-PRP on human fibroblast proliferation in vitro.
METHODS
LP-PRP was prepared from 49 donors. The fibroblasts were seeded, and at 24 hours after seeding, 1 × 107/10 µL LP-PRP was added once to each well. The cells were harvested 10 times during study period at our planned points, and we examined cell proliferation using the water-soluble tetrazolium salt-1 assay. We collected the supernatants and measured the amount of growth factors such as platelet-derived growth factor (PDGF)-AB/BB, insulin-like growth factor-1 (IGF-1), transforming growth factor-β1 (TGF-β1), and vascular endothelial growth factor (VEGF), which are known to be involved in wound healing processes, by multiplex assay.
RESULTS
Human fibroblasts treated with LP-PRP showed a significant increase in proliferation when compared to untreated controls (p < 0.001 at days 4, 6, and 8). Multiplex cytokine assays revealed various secretion patterns. PDGF-AB/BB appeared at early time points and peaked before fibroblast proliferation. IGF-1 and TGF-β1 secretion gradually increased and peaked on days 4 and 6 post-treatment. The early VEGF concentration was lower than the concentration of other growth factors but increased along with cell proliferation.
CONCLUSIONS
Platelets in LP-PRP release growth factors such as PDGF, IGF-1, TGF-β1 and VEGF, and these growth factors have a promoting effect for human fibroblast proliferation, one of the important mediators of wound healing. These results suggest that growth factors derived from LP-PRP enhance the proliferation of human fibroblast.

Keyword

Fibroblast; Platelet-rich plasma; Platelet-derived growth factor; Transforming growth factor-β1

MeSH Terms

Cell Proliferation
Fibroblasts*
Humans*
In Vitro Techniques*
Insulin-Like Growth Factor I
Intercellular Signaling Peptides and Proteins*
Platelet-Derived Growth Factor
Platelet-Rich Plasma
Soft Tissue Injuries
Tissue Donors
Vascular Endothelial Growth Factor A
Wound Healing
Insulin-Like Growth Factor I
Intercellular Signaling Peptides and Proteins
Platelet-Derived Growth Factor
Vascular Endothelial Growth Factor A

Figure

• 

  Fig. 1 Leukocyte-poor platelet-rich plasma (LP-PRP) Arthrex ACP platelet separation system. Blood (13.5 mL) and anticoagulant citrate dextrose solution A (ACD-A, 1.5 mL) were mixed and transferred into a double ACP syringe and centrifuged at 380 ×g (1,500 rpm) for 5 minutes to separate the red blood cell/leukocyte and plasma-platelet layers, the latter of which was collected as LP-PRP (about 4 mL).

• 

  Fig. 2 (A) Human fibroblast culture and proliferation assay images of fibroblast cultures before and after leukocyte-poor platelet-rich plasma (LP-PRP) treatment. (B) Cell proliferation was examined at the indicated time points by water-soluble tetrazolium salt-1 assay. The LP-PRP-treated group showed the most robust growth on 4, 6, and 8 days. We performed this experiment in triplicate and three times. Values are presented as mean ± standard deviation of triplicate measures in three independent experiments. *p < 0.001.

• 

  Fig. 3 Growth factor concentrations in leukocyte-poor platelet-rich plasma (LP-PRP) platelet-derived growth factor (PDGF)-AB/BB (A), insulin-like growth factor-1 (IGF-1) (B), transforming growth factor-β1 (TGF-β1) (C), and vascular endothelial growth factor (VEGF) (D) were monitored at the indicated time intervals. Values are presented as mean ± standard deviation of triplicate measures in three independent experiments. *p < 0.001.

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