Liquid-liquid phase transition as a basis for novel materials for skin repair and regeneration.

ABSTRACT

Inorganic materials are of increasing interest not only for bone repair but also for other applications in regenerative medicine. In this study, the combined effects of energy-providing, regeneratively active inorganic polyphosphate (polyP) and also morphogenetically active pearl powder on wound healing were investigated. Aragonite, the mineralic constituent of pearl nacre and thermodynamically unstable form of crystalline calcium carbonate, was found to be converted into a soluble state in the presence of a Ca2+-containing wound exudate, particularly upon addition of sodium polyP (Na-polyP), driven by the transfer of Ca2+ ions from aragonite to polyP, leading to liquid-liquid phase separation to form an aqueous Ca-polyP coacervate. This process is further enhanced in the presence of Ca-polyP nanoparticles (Ca-polyP-NP). Kinetic studies revealed that the coacervation of polyP and nacre aragonite in wound exudate is a very rapid process that results in the formation of a stronger gel with a porous structure compared to polyP alone. Coacervate formation, enabled by phase transition of crystalline aragonite in the presence of Na-polyP/Ca-polyP-NP and wound exudate, could also be demonstrated in a hydroxyethyl cellulose-based hydrogel used for wound treatment. Furthermore, it is shown that Na-polyP/Ca-polyP-NP together with nacre aragonite strongly enhances the proliferation of mesenchymal stem cells and promotes microtube formation in the in vitro angiogenesis assay with HUVEC endothelial cells. The latter effect was confirmed by gene expression studies, applying real-time polymerase chain reaction, using the biomarker genes VEGF (vascular endothelial growth factor) and hypoxia-inducible factor-1 α (HIF-1α). Division of Escherichia coli is suppressed when suspended in a matrix containing Na-polyP/Ca-polyP-NP and aragonite. The potential medical relevance of these findings is supported by an animal study on genetically engineered diabetic mice (db/db), which demonstrated a marked increase in granulation tissue and microvessel formation in regenerating experimental wounds treated with Ca-polyP-NP compared to controls. Co-administration of aragonite significantly accelerated the wound healing-promoting effect of polyP in db/db mice. Based on these results, we propose that the ability of polyP to form a mixed coacervate with aragonite, in addition to its energy (ATP)-generating function, can decisively contribute to the regenerative activity of this polymer in wound repair.