Method to obtain a plasma rich in platelet- and plasma-growth factors based on water evaporation

Abstract

Platelet-Rich Plasma, also known as PRP, is an autologous biologic product used in medicine as a treatment for tissue repair. Nowadays, the majority of PRP obtention methods enrich only platelets, not considering extraplatelet biomolecules, which take part in several cell processes. In the present work, a novel PRP preparation method was developed to obtain a PRP rich in both platelet and plasma extraplatelet molecules. The method is based on the evaporation of the water of the plasma using a rotary evaporator. With this new methodology an increase in plasmatic growth factors and, as a consequence, a better dermal fibroblast cell viability was achieved, compared to a standard PRP formulation. This novel PRP product obtained with this new methodology showed promising results in vitro as an improved PRP treatment in future application.

Fig 1. Schematic representation of the process to obtain nPRP using a rotary evaporator.

PLT 1X represents similar levels of platelets comparing to basal levels, while TP 1X represents total proteins at basal levels. After the evaporation, both PLT and TP are concentrated, doubling its concentration.

Fig 2. Platelet and total protein concentration levels in standard and novel PRP.

Fold change values of (A) platelets and (B) proteins in blood and standard and novel PRP are shown. Error bars = standard deviation of eight donors. Statistically significant differences were calculated by Kruskall-Wallis and Dunn’s multiple comparison test post hoc for the platelet content and one-way ANOVA and Tukey’s multiple comparison test post hoc for total protein content (*** p<0.001; **** p<0.0001).

Fig 3. nPRP clotting time subject to sample pH.

Different pH values are represented considering the pH of the nPRP. From pH 8.6, and after decreasing the sample to pH 8 and to physiological values with HCl solution. Values expressed as median ± 95% CI of eight donors. For the statistics, Kruskal-Wallis test was done with Dunn’s multiple comparison post hoc (****p < 0.0001).

Fig 4. Platelet activation in standard and novel PRP.

The graph represents the percentage of positive cells that are indicative of activated platelets (resting condition) and platelets activated by adding ADP as a platelet activator (stimulate condition). Values expressed as mean ± standard deviation of eight donors. Statistical significance was calculated by two-way ANOVA (**p < 0.01; ****p < 0.0001).

Fig 5. Growth factor levels presented in blood, sPRP and nPRP.

Platelet (A) PDGF and plasmatic (B) HGF and (C) IGF-1 growth factor concentration levels are expressed as mean ± standard deviation of eight donors. Statistical significance was calculated by one-way ANOVA and Tukey’s multiple comparison test post hoc (*p < 0.05 and ***p < 0.001).

Fig 6. NHDF cell proliferation analysis.

The viability levels of the cells incubated with sPRP and nPRP are expressed as relative lights units (RLU) and each point represents a different donor (n = 8). Statistical analysis was calculated by t-test (** p < 0.01).