The regenerative effect of Platelet Rich Plasma on skin and other tissue lesions is well known. If, on the one hand, research aims to optimize PRP standardized protocols, on the other hand, it aims to identify substrates as vehicles for the platelet content release to the lesion site. For the latter purpose, hyaluronic acid (HA) is proposed thanks to its viscoelastic and biological properties and biocompatibility. The aim of this study was the set-up and characterization of an “off the shelf” freeze-dried and injectable device based on HA that entraps PRP in a stable matrix sustaining the platelet growth factor release.
High MW HA (1400 KDa – HA-HMW) and Low MW HA (56, 90, 200 KDa – HA-LMW) were used in combination with PRP at the platelet concentration of 2.5x106 plts/ul in the ratio 1:1. The Rheology of selected HAs with defined hydrodynamic parameters was analyzed at the concentration used for PRP mixing and lyophilization. After regeneration, the resulting lyophilized mixtures HA/PRP were tested for in vitro cell proliferation and scratch assays on human primary fibroblasts. The biological activity of freeze-dried HA-LMW/PRP formulations were also tested during storage at different temperatures (25°C, 4°C and -20°C) up to 6 months.
In a first set of experiments, HA-HMW/PRP was evaluated for its biological activity, showing that the freeze-dried and regenerated HA-PRP combination supported human dermal fibroblast proliferation in a comparable way to PRP alone. Although the biological properties of the HA-HMW/PRP were maintained, the formulation needed almost half an hour for full regeneration and quite a strong pressure to be extruded by a 21-gauge needle.
To overcome these limitations, hyaluronans of low molecular weight were selected after a specific hydrodynamic (SEC-TDA) analysis, namely HA 56 KDa, HA 90 KDa, HA 200 KDa. All formulations obtained by the combination of these HA with PRP induced cell proliferation. For a clinical application of an “off the shelf” lyophilized product it is mandatory to preserve the PRP activity along time. We already reported that the long-term storage of the freeze-dried PRP was associated to a progressive biological activity loss. In this work, the HA/PRP formulation were tested to evaluate the possible stabilization by HA at different temperatures and length of storage.
All formulations induced cell proliferation comparable to PRP alone at the different tested temperatures, but, interestingly, the 56 KDa HA/PRP formulation, after 6 months of storage at 25°C, showed significant preservation of the proliferation activity compared to PRP alone, suggesting a protective effect of HA versus the PRP bioactive factors. In addition, some of the low molecular weight HA-PRP formulation showed, at the same storage condition, a superior healing rate in a scratch assay followed by time lapse video microscopy.
In conclusion, we developed a lyophilized HA-based/PRP device, that may improve bioadhesive properties of the sole PRP also improving on site delivering (e.g. wound treatment). These formulations proved to release platelet factors preserving their biological activity over time. HA/PRP allows the development of promising products, for topical and intra-articular applications."