*3.1. PRP Prevented TGF-*β*1- Induced Fibroblast to Myofibroblast Transition*

Successful in vitro differentiation of NIH/3T3 fibroblasts towards myofibroblasts induced by the well-known pro-fibrotic factor TGF-β1 and the ability of PRP to prevent this transition were confirmed by morphological, biochemical and electrophysiological evaluations. Fibroblasts induced to differentiate by culturing in DM exhibited the typical features of myofibroblastic phenotype. Indeed, as judged by Western blotting analysis, they showed a significant increase of the expression of α-sma (*p* < 0.05), the most reliable marker of myofibroblasts, after 48 h and even more after 72 h of culture, as compared to control undifferentiated cells in PM (Figure 1A,B). Moreover, the immunocytochemical analysis at confocal microscopy, performed after 72 h of culture, confirmed the data of Western blotting and showed that this protein was well organized along filamentous structures (Figure 1C,D,I).

**Figure 1.** Evaluation of the effects PRP on fibroblast to myofibroblast transition and of the involvement of GJs: α-sma expression. Fibroblasts were induced to differentiate into myofibroblasts by culturing in differentiation medium (DM) in the presence or absence of PRP for 48 h and 72 h. Cells cultured in proliferation medium (PM) served as control undifferentiated cells. In parallel experiments, fibroblasts were cultured in PM or in DM in the presence of heptanol (HEPT), a common GJ channel blocker, in the presence or absence of PRP for 72 h. (**A**,**B**) Western Blotting analysis of α-sma expression. (**A**) Representative Blot. (**B**) Histogram showing the densitometric analysis of the bands normalized to α-tubulin. (**C**–**H**) Representative confocal fluorescence images of the cells immunostained with antibodies against α-sma (green) and counterstained with propidium iodide (PI) to detect nuclei. Scale bar: 50 μm. (**I**) Histogram showing the densitometric analysis of the intensity of the α-sma fluorescence signal performed on digitized images in 20 regions of interest (ROI) of 100 μm2 for each confocal stack (10). Data shown are mean ± S.E.M. and represent the results of at least three independent experiments performed in triplicate. Significance of difference: \* *p* < 0.05 versus PM; ◦ *p* < 0.05 versus DM 48 h; # *p* < 0.05 versus DM 72 h; § *p* < 0.05 versus DM + PRP 48 h; & *p* < 0.05 versus DM + PRP 72 h; \$ *p* < 0.05 versus PM + HEPT 72 h (One-way ANOVA followed by the Tukey post hoc test).

Moreover, cells cultured in DM for 72 h, appeared much larger with a more polygonal shape as compared to the cells cultured in PM which, instead, were smaller and spindle-shaped as judged by the confocal fluorescence analysis after labeling with the membrane dye Alexa Fluor 488 conjugated WGA (Figure 2A,B). Differentiated cells also showed a robust increase (*p* < 0.05) in the expression of type-1 collagen at the cytoplasmic level and, in some cases, even outside the cells in a filamentous form (Figure 2D,E,G).

**Figure 2.** Effects of PRP on fibroblast to myofibroblast transition: Cell morphology and type-1 collagen expression. Fibroblasts were induced to differentiate into myofibroblasts by culturing in differentiation medium (DM) in the presence or absence of PRP for 72 h. The cells cultured in proliferation medium (PM) served as control undifferentiated cells. (**A**–**F**) Representative confocal fluorescence images of the cells (**A**–**C**) stained with Alexa Fluor 488-conjugated WGA (green) to reveal the plasma membrane and (**D**–**F**) immunostained with antibodies against type-1 collagen (green) and counterstained with propidium iodide (PI), to label nuclei. Scale bar: 50 μm. (**G**) Histogram showing the densitometric analysis of the intensity of type-1 collagen fluorescence signal performed on digitized images in 20 regions of interest (ROI) of 100 <sup>μ</sup>m2 for each confocal stack (10). Data are reported as mean <sup>±</sup> S.E.M. and represent the results of at least three independent experiments performed in triplicate. Significance of difference: \* *p* < 0.05 versus PM; ◦ *p* <0.05 versus DM (One-way ANOVA followed by the Tukey post hoc test).

The electrophysiological analysis of the passive membrane properties achieved by the whole-cell patch-clamp technique confirmed that the cells cultured in DM acquired the myofibroblastic phenotype. First, the resting membrane potential, RMP, was recorded and it was found that the values recorded from

the cells cultured in DM for 48 h tended to be more depolarized compared to control undifferentiated fibroblasts in PM, in accordance with previous observations [23,24]. The overall results from all of the experiments done are shown in Figure 3A and Table 1. The statistical analysis of the RMP values between the different conditions was achieved with one–way ANOVA that provided overall results for our data. Despite the observed tendency to depolarization, the differences between the means did not turned out to be statistically significant (*p* = 0.26; F = 1.45 < Fcrit = 3.15; df = 30).

We then analyzed the cell membrane resistance, Rm, in the voltage-clamp mode of our device. As shown in Figure 3B, Rm increased after 48 h and even more after 72 h of culture in DM. The one-way ANOVA analysis of Rm indicated statistical significance (*p* = 0.00010; F = 8.99 > Fcrit = 2.83; df = 50). To know which groups were significantly different from another, we used the Bonferroni post-hoc test. The resulting significance is indicated by the symbols depicted in Figure 3B and Table 1.

**Figure 3.** Effects of PRP on fibroblast to myofibroblast transition: Electrophysiological analysis of biophysical properties. (**A**) Resting membrane potential (RMP, in mV) recorded in the different conditions. Myofibroblasts have a tendency to be more depolarized. (**B**) Membrane resistance (Rm, in MΩ) shows higher values in myofibroblasts grown in differentiation medium (DM) compared to fibroblasts grown in proliferation medium (PM). (**C**) Membrane capacitance (Cm, in pF): Myofibroblasts show higher values compared to the undifferentiated cells in PM. All values are reported as mean ± S.E.M. and are listed in Table 1. \* *p* < 0.05 versus PM; # *p* < 0.05 versus DM + PRP 48 h; § *p* < 0.05 versus DM 48 h (one-way ANOVA, followed by Bonferroni's post hoc test).


**Table 1.** Electrophysiological analysis of the membrane passive properties.

Data are reported as mean ± S.E.M. \* *<sup>p</sup>* <sup>&</sup>lt; 0.05 versus PM; # *<sup>p</sup>* <sup>&</sup>lt; 0.05 versus DM <sup>+</sup> PRP 48 h; § *<sup>p</sup>* <sup>&</sup>lt; 0.05 versus DM 48 h (one-way ANOVA, followed by Bonferroni's post hoc test). The number of investigated cells is indicated by "*n*" in brackets for each condition.

Similarly, the cell capacitance, Cm, of cells cultured in DM, usually assumed as an index of cell surface, changed significantly (*p* = 0.0085; F = 4.52 > Fcrit = 2.86; df = 45; one way ANOVA). It tended to increase compared to that estimated in PM (Figure 3C; Table 1), being (*p* < 0.05) higher for cells in DM, especially after 48 h. These results were consistent with the observed cell morphology (Figure 2A,B).

The treatment with PRP actually counteracted the TGF-β1-induced fibroblast to myofibroblast transition. Indeed, the cells cultured in DM + PRP exhibited a significant (*p* < 0.05) reduction of α-sma (Figure 1A,B,E,I) with respect to differentiated cells in DM, together with different morphology, more similar to that of cells cultured in PM (Figure 2C) and a significantly reduced expression of type-1 collagen (*p* < 0.05) (Figure 2F,G).

Of interest, the electrophysiological analyses performed on the cells cultured in DM + PRP for the first time, revealed that Rm values tended to decrease compared to those measured in DM (*p* > 0.05) both after 48 h and 72 h of culture (Figure 3B; Table 1). As well, Cm values evaluated from cells cultured in DM + PRP were significantly reduced (*p* < 0.05) compared to those in DM, consistent with the observed changed morphology of these cells (Figure 3C; Table 1).
