Platelet concentration in platelet-rich plasma affects tenocyte behavior in vitro

Abstract

Since tendon injuries and tendinopathy are a growing problem, sometimes requiring surgery, new strategies that improve conservative therapies are needed. Platelet-rich plasma (PRP) seems to be a good candidate by virtue of its high content of growth factors, most of which are involved in tendon healing. This study aimed to evaluate if different concentrations of platelets in PRP have different effects on the biological features of normal human tenocytes that are usually required during tendon healing. The different platelet concentrations tested (up to 5 × 10(6) plt/µL) stimulated differently tenocytes behavior; intermediate concentrations (0.5 × 10(6), 1 × 10(6) plt/µL) strongly induced all tested processes (proliferation, migration, collagen, and MMPs production) if compared to untreated cells; on the contrary, the highest concentration had inhibitory effects on proliferation and strongly reduced migration abilities and overall collagen production but, at the same time, induced increasing MMP production, which could be counterproductive because excessive proteolysis could impair tendon mechanical stability. Thus, these in vitro data strongly suggest the need for a compromise between extremely high and low platelet concentrations to obtain an optimal global effect when inducing in vivo tendon healing.

Figure 1

Tenocytes characterization. Tenocytes were characterized through vimentin immunostaining (a) and assay of scleraxis expression by western blot (b). Magnification 200x.

Figure 2

Platelet-gel-released supernatant stimulates tenocyte proliferation. The effects of different PG concentrations on tenocyte proliferation after 72, 96, and 120 h. The value obtained from untreated cells (CTRL−) was considered to be 100% proliferation. White bars (CTRL+) refer to cells grown in complete medium (positive control). Data originated in triplicate (n = 3) and were analyzed by two-way ANOVA, followed by Dunnett test, *** = P < 0.001. Error bars correspond to standard deviation.

Figure 3

Morphological analysis of PG-supernatant-treated tenocytes. (a) Positive control (cells grown in complete medium); (b) negative control (cells grown in medium + 1% FBS); (c) cells treated with 0.5 × 10⁶ plt/μL; (d) cells treated with 1 × 10⁶ plt/μL; (e) cells treated with 2 × 10⁶ plt/μL; and (f) cells treated with 3 × 10⁶ plt/μL. Magnification 100x.

Figure 4

The effect of platelet-gel-released supernatant on tenocyte migration. A summary panel presenting the effects of different concentrations (rows) of platelet-gel-released supernatant on wound healing after 22, 30, and 46 h (columns). Image at 0 h is representative of the starting situation of all conditions. Magnification 100x.

Figure 5

Gelatinases assay. Gelatin zymography showing the effects of different platelet-gel-released supernatant concentrations on gelatinases (MMP-2 and MMP-9) production. The figure was cropped removing upper and lower parts of gel, which contained no bands. Lane 1: positive control (cells grown in complete medium). Lane 2: negative control (cells grown in medium + 1% FBS). Lane 3: cells treated with 0.5 × 10⁶ plt/μL. Lane 4: cells treated with 1 × 10⁶ plt/μL. Lane 5: cells treated with 2 × 10⁶ plt/μL. Lane 6: cells treated with 3 × 10⁶ plt/μL. The table shows the densitometric values expressed as % volume of pro-MMP-9 and pro-MMP-2. For the pro-MMP-9 densitometric analysis the band of cells that were treated with 0.5 × 10⁶ plt/μL was set at 100%, and for the pro-MMP-2 densitometric analysis the band of untreated cell (negative control) was set at 100%.

Figure 6

The effect of platelet-gel-released supernatant on collagen release. The effects of different PG-released supernatant concentrations on type I collagen release. Lane 1: positive control (cells grown in complete medium). Lane 2: negative control (cells grown in medium + 1% FBS). Lane 3: cells treated with 0.5 × 10⁶ plt/μL. Lane 4: cells treated with 1 × 10⁶ plt/μL. Lane 5: cells treated with 2 × 10⁶ plt/μL. Lane 6: cells treated with 3 × 10⁶ plt/μL. The table shows the densitometric values (expressed as % volume; band of negative control was set to 100%) of the pro-α1 and α2 collagen subtypes.

Figure 7

Platelet-gel-released supernatant affects scleraxis expression. The effects of different PG-released supernatant concentrations on scleraxis expression. The figures were cropped for more clarity; the removed areas contained no bands. Lane 1: negative control (cells grown in medium + 1% FBS). Lane 2: positive control (cells grown in complete medium). Lane 3: cells treated with 0.5 × 10⁶ plt/μL. Lane 4: cells treated with 1 × 10⁶ plt/μL. Lane 5: cells treated with 2 × 10⁶ plt/μL. Lane 6: cells treated with 3 × 10⁶ plt/μL. Actin detection was utilized as loading control. Values from densitometric analysis are shown on base of each protein band and were calculated as described in the Materials and Methods section.