The effects of PRP (A) and HGF (B) treatment on COX-1 and COX-2 protein expression in tendon cell culture.

<p>Total proteins extracted from tendon cells after treatments were separated on 12% SDS-PAGE and transferred onto nitrocellulose membrane to detect COX-1 and COX-2 proteins using goat anti-COX-1 and goat anti-COX-2 polyclonal antibodies, respectively, and peroxidase-conjugated rabbit anti-goat IgG antibody as secondary antibody. <b>A.</b> PRP treatment. Lane 1: serum free medium (SF); lane 2: IL-1β (1 ng/ml); lane 3: PRP (2%)+IL-1β (1 ng/ml); lane 4: PRP (10%)+ IL-1β (1 ng/ml); lane 5: PRP (2%)+ IL-1β (1 ng/ml)+HGF antibody (10 ng/ml); lane 6: PRP (10%)+IL-1β (1 ng/ml)+HGF antibody (10 ng/ml). Notice that 10% PRP reduced COX-1 expression, but almost completely inhibited COX-2 expression (lane 4 vs. lane 2). <b>B.</b> HGF treatment. Lane 1: SF; lane 2: IL-1β (1 ng/ml); lane 3: HGF (1 ng/ml)+IL-1β (1 ng/ml); lane 4: HGF (1 ng/ml); and lane 5: HGF (1 ng/ml)+IL-1β (1 ng/ml)+HGF antibody (10 ng/ml). Also notice that HGF at 1 ng/ml markedly reduced both COX-1 and COX-2 expression (lane 3 vs. lane 2).</p

Immunostaining of COX-1 and COX-2 on tissue sections of wounded mouse Achilles on day 3 after wounding and PRP treatment.

<p>Dissected tissues were cut, fixed in 4% paraformaldehyde. For COX-1, the tissue sections were stained with goat anti-COX-1 antibody and FITC-conjugated rabbit anti-goat IgG antibody. For COX-2, the tissue sections were stained with goat anti-COX-2 antibody and Cy-3 conjugated donkey anti-goat IgG antibody. Green represents COX-1; red represents COX-2; and blue represents nuclei stained with Hoechst 33342. Both COX-1 (<b>A</b>) and COX-2 (<b>B</b>) were positively stained on the wound sites of the mouse tendons without PRP treatment. However, treatment with PRP, with its platelet concentration about 4 times over that of mouse whole blood (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067303#pone-0067303-g009" target="_blank"><b>Figure </b><b>9</b></a>), nearly abolished COX-1 (<b>C</b>) and COX-2 (<b>D</b>) expressions. Bar: 100 µm.</p

The effects of PRP or HGF treatment on COX-1 protein expression in tendon cell culture by immunostaining.

<p>Tendon cells in culture were treated with IL-1β, PRP, HGF, and/or HGF antibody (AB) to determine COX-1 expression using goat anti-COX-1 antibody and Cy-3 conjugated donkey anti-goat IgG antibody. Red represents COX-1 protein; and blue represents nuclei stained with Hoechst 33342. IL-1β treatment induced a high level of COX-1 expression in tendon cells compared to the control conditions (<b>A</b>); however, such an increase in COX-1 was markedly reduced by PRP or HGF treatments. The addition of HGF antibody to tendon cell cultures decreased the reduction by PRP or HGF treatments. <b>A:</b> SF (control conditions); <b>B:</b> IL-1β (1 ng/ml); <b>C:</b> 2%PRP+ IL-1β; <b>D:</b> 10%PRP+IL-1β; <b>E:</b> HGF+IL-1β; <b>F:</b> 2%PRP+IL-1β+AB; <b>G:</b> 10%PRP+IL-1β+AB; <b>H:</b> HGF+IL-1β+AB; <b>I:</b> 2%PRP; <b>J:</b> 10%PRP; <b>K:</b> HGF (1 ng/ml) and <b>L:</b> HGF+AB (10 ng/ml). Bar: 100 µm.</p

The effects of PRP (A) and HGF (B) treatment on COX-2 gene expression in tendon cell culture.

<p>Both PRP and HGF treatments nearly abolished COX-2 expression, which was induced by IL-1β treatment of tendon cells. Note that *P<0.05 is with respect to IL-1β treatment condition (<b>I</b>); and ^#P<0.05 is with respect to the PRP+IL-1β (<b>P+I</b>) group in <b>A</b> and the HGF+IL-1β (<b>H+I</b>) group in <b>B</b>. The data are expressed as mean ± SD, n  = 4. Label abbreviations and concentrations of all agents (IL-1β, PRP, HGF, and HGF antibody) are identical to legends in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067303#pone-0067303-g002" target="_blank"><b>Figure 2</b></a>. For SD values that are barely visible, the variation in gene expression values was less than 5 (control group, SF is 1).</p

Immunostaining of COX-1 and COX-2 on tissue sections of wounded mouse Achilles tendons on day 3 after wounding and HGF treatment.

<p>Mouse Achilles tendons were dissected 3 days after wounding and with or without HGF treatment followed by mincing and fixing tissues in 4% paraformaldehyde. For COX-1 and COX-2 staining, the tissue sections were stained with goat anti-COX-1 and goat anti-COX-2 antibodies respectively, and detected using FITC-conjugated rabbit anti-goat IgG antibody (COX-1) or Cy-3 conjugated donkey anti-goat IgG antibody (COX-2) respectively. Green represents COX-1; red represents COX-2; and blue represents nuclei stained with Hoechst 33342. Both COX-1 (<b>A</b>) and COX-2 (<b>B</b>) were positively stained on the wounded area without HGF treatment. However, HGF treatment (3 ng in 10 µl saline) decreased COX-1 (<b>C</b>) and COX-2 (<b>D</b>) expression. Bar: 100 µm.</p

Platelet numbers in mouse whole blood, PRP, and PPP preparations used for the <i>in vivo</i> experiments.

<p>On average, the number of platelets in PRP preparations was about four times higher than in mouse whole blood (WB), whereas the number was 60 times higher in PRP than in PPP preparations (*P<0.05 is with respect to WB). The data are expressed as mean ± SD, and n  = 8.</p

The effects of PRP (A) and HGF (B) treatments on PGE₂ production in tendon cell culture.

<p>IL-1β treatment (<b>I</b>) induced high levels of PGE₂ production in cells compared to levels induced by control conditions (<b>SF</b>); however, these increases in PGE₂ were markedly reduced by PRP or HGF treatment. Note that *P<0.05 is with respect to IL-1β treatment condition (<b>I</b>); and ^#P<0.05 is with respect to the PRP+IL-1β (<b>P+I</b>) group in <b>A</b> and the HGF+IL-1β (<b>H+I</b>) group in <b>B</b>. The data are expressed as mean ± SD, n  = 4. Label abbreviations and concentrations of all agents (IL-1β, PRP, HGF, and HGF antibody) are identical to legends in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067303#pone-0067303-g002" target="_blank"><b>Figure 2</b></a>. For SD values that are barely visible, the variation in gene expression values was less than 5 (control group, SF is 1).</p

The effect of PRP (A) and HGF (B) treatment on COX-1 expression in tendon cell culture.

<p>Tendon cells were treated with IL-1β, which resulted in high levels of COX-1 expression; however, the addition of PRP (<b>A</b>) or HGF (<b>B</b>) together with IL-1β to the cell culture markedly reduced the level of COX-1 expression. Also, PRP or HGF alone did not alter COX-1 expression. Note that *P<0.05 is with respect to IL-1β treatment (<b>I</b>); and ^#P<0.05 is with respect to the PRP+IL-1β (<b>P+I</b>) group in <b>A</b> and the HGF+IL-1β (<b>H+I</b>) group in <b>B</b>. The data are expressed as mean ± SD, n  = 4. <b>SF:</b> serum free medium; <b>I:</b> IL-1β; <b>P+I:</b> PRP+IL-1β; <b>P+AB+I:</b> PRP+HGF antibody+IL-1β; <b>P:</b> PRP; <b>P+AB:</b> PRP+HGF antibody; <b>H+I:</b> HGF+ IL-1β; <b>H+AB+I:</b> HGF+HGF antibody+IL-1β, <b>H:</b> HGF; and <b>H+AB:</b> HGF+HGF antibody. Concentrations of the reagents used were: IL-1β, 1 ng/ml; PRP, 10% (v/v); HGF, 1 ng/ml; HGF antibody, 10 ng/ml. For SD values that are barely visible, the variation in gene expression values was less than 5 (control group, SF is 1).</p

HGF levels in rabbit whole blood and PRP preparations.

<p>The concentration of HGF in PRP was over 3.5 times the concentration found in rabbit whole blood (WB). The data are expressed as mean ± SD, n  = 7.</p

The effects of PRP (A) and HGF (B) treatment on mPGES-1 gene expression in tendon cell culture.

<p>IL-1β treatment increased mPGES-1 expression (<b>I</b>), but its effect was almost completely suppressed by PRP (<b>A</b>) or HGF (<b>B</b>) treatment. Note that *P<0.05 is with respect to IL-1β treatment condition (<b>I</b>); and ^#P<0.05 is with respect to the PRP+IL-1β (<b>P+I</b>) group in <b>A</b> and the HGF+IL-1β (<b>H+I</b>) group in <b>B</b>. The data are expressed as mean ± SD, n  = 4. Label abbreviations and concentrations of all agents (IL-1β, PRP, HGF, and HGF antibody) are identical to legends in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067303#pone-0067303-g002" target="_blank"><b>Figure 2</b></a>. For SD values that are barely visible, the variation in gene expression values was less than 5 (control group, SF is 1).</p