Platelet-rich plasma modulates the secretion of inflammatory/angiogenic proteins by inflamed tenocytes

Abstract

Background: Platelet-rich plasma therapies for tendinopathy appear to provide moderate pain reduction. However, the biological mechanisms behind the observed clinical effects remain poorly characterized.

Questions/purposes: The purpose of this study was to explore whether platelet-rich plasma modifies the inflammatory/angiogenic status of already inflamed tenocytes by examining (1) gene expression; (2) modulation of chemokine and interleukin secretion; and (3) differences between healthy and tendinopathic tenocytes.

Methods: Cells from both healthy and tendinopathic tendons were exposed to interleukin (IL)-1ß and after treated with platelet-rich plasma. Modifications in the expression of selected genes were assessed by real-time reverse transcription-polymerase chain reaction and changes in secretion of angiogenic/inflammatory molecules by enzyme-linked immunosorbent assay. Platelet-rich plasma-induced changes in tendinopathic cells were compared with normal after normalizing platelet-rich plasma data against IL-1ß status in each specific sample.

Results: In IL-1ß-exposed cells, platelet-rich plasma downregulates expression of IL-6/CXCL-6 (mean, 0.015; 95% confidence interval [CI], 0.005-0.025; p = 0.026), IL-6R (mean, 0.61; 95% CI, 0.27-0.95; p = 0.029), and IL-8/CXCL-8 (mean, 0.02; 95% CI, 0.007-0.023; p = 0.026). Secretion of IL-6/CXCL6, 0.35 (95% CI, 0.3-0.4; p = 0.002), IL-8/CXCL8, 0.55 (95% CI, 0.5-0.7; p = 0.01), and monocyte chemoattractant protein-1/CCL2, 0.40 (95% CI, 0.2-0.6; p = 0.001) was reduced by platelet-rich plasma, whereas vascular endothelial growth factor increased by twofold, (95% CI, 1.7-2.3; p < 0.001). RANTES/CCL5 increased by10-fold (95% CI, 4-17) and hepatocyte growth factor by 21-fold (95% CI, 0.2-42) in tendinopathic and by 2.3-fold (95% CI, 2-3) and threefold (95% CI, 1-5) in normal cells (p = 0.005 for both).

Conclusions: Platelet-rich plasma induces an immunomodulatory and proangiogenic phenotype consistent with healing mechanisms with few differences between tendinopathic and normal cells.

Clinical relevance: Platelet-rich plasma injections in pathological and nearby tissue might help to recover tendon homeostasis.

Figures

Fig. 1

Experimental design: after cell-cycle synchronization (30 hours using 0.2% PRP), starved cells (t = 0 hours) were treated with 1 ng/mL rh-IL-1ß for 2 days and then exposed to 10% PRP for the next 3 days. Experiments were performed in parallel with healthy tendon cells from the semitendinosus tendon (N = 2 donors) and tendinopathic tendon cells from the rotator cuff (N = 2 donors). PRPs were prepared from four healthy donors and used to treat healthy cells and from four patients with tendinopathy and used to treat tendinopathic cells. Both the tendon cells and cell culture supernatants were harvested in three different situations: (1) after starvation (constitutive secretion); (2) some cells were starved and harvested after 48-hour exposure to IL-1ß (inflamed cells); and (3) other cells received both IL-1 (for 48 hours) and an additional 72-hour PRP exposure (PRP-treated cells). RT-PCR = reverse transcription-polymerase chain reaction.

Fig. 2A–D

Relative gene expression of (A–B) modulators of inflammation and (C–D) extracellular matrix proteins. Boxes illustrate the relative mRNA expression; the band inside the box is the median. mRNA folds of PRP-treated tendon cells are calculated relative to tendon cells subjected to inflammatory stimuli (IL-1ß).

Fig. 3A–B

Secretion of inflammatory/angiogenic molecules relative to IL-1ß exposed tendon cells. (A) Tendon cells treated with PRP showed a reduction in the secretion of IL-6, IL-8, and MCP-1 but did not show changes in the secretion of GRO-alpha. (B) Tendon cells treated with PRP showed increased secretion of VEGF, HGF, RANTES, and GAG but no changes in PGE2 secretion.

Fig. 4A–C

Similarities and differences in protein secretion by healthy tendon cells and cells from tendinopathic tissue. PRP-induced changes in tendinopathic were compared with normal after normalizing PRP data against IL-1β status in each specific sample. Chemokine secretion is not modified by PRP (A). Differences were found in RANTES (B) and HGF secretion (C).