No abstract

Inorganic polyphosphate [polyP] has proven to be a promising physiological biopolymer for potential use in regenerative medicine because of its morphogenetic activity and function as an extracellular energy-donating system. Amorphous Ca -polyP nanoparticles [Ca-polyP-NPs] are characterized by a high zeta potential with -34 mV (at pH 7.4). This should contribute to the stability of suspensions of the spherical nanoparticles (radius 94 nm), but make them less biocompatible. The zeta potential decreases to near zero after exposure of the Ca-polyP-NPs to protein/peptide-containing serum or medium plus serum. Electron microscopy analysis reveals that the particles rapidly change into a coacervate phase. Those mats are amorphous, but less stable than the likewise amorphous Ca-polyP-NPs and are morphogenetically active. Mesenchymal stem cells grown onto the polyP coacervate show enhanced growth/proliferation and become embedded in the coacervate. These results suggest that the Ca-polyP coacervate, formed from Ca-polyP-NPs in the presence of protein, can act as an adaptable framework that mimics a niche and provides metabolic energy in bone/cartilage engineering.

Here, we studied the potential role of inorganic polyphosphate (polyP) as an energy source for ADP and ATP formation in the extracellular space. In SaOS-2 cells, we show that matrix vesicles are released into the extracellular space after incubation with polyP. These vesicles contain both alkaline phosphatase (ALP) and adenylate kinase (AK) activities (mediated by ALPL and AK1 enzymes). Both enzymes translocate to the cell membrane in response to polyP. To distinguish the process(es) of AMP and ADP formation during ALP hydrolysis from the ATP generated via the AK reaction, inhibition studies with the AK inhibitor A(5')P5(5')A were performed. We found that ADP formation in the extracellular space occurs after enzymatic ATP synthesis. After exposure to polyP, a significant increase of the ADP level was observed, which is likely to be been catalyzed by ALP. This increase is not due to an intensified ATP release via exocytosis. The ATP level in the extracellular space of SaOS-2 cells is strongly increased in response to polyP, very likely mediated by the AK. We propose that the ALP and AK enzymes are involved in the extracellular ADP and ATP synthesis.

We investigated the effect of bioglass (bioactive glass) on growth and mineralization of bone-related SaOS-2 cells, encapsulated into a printable and biodegradable alginate/gelatine hydrogel. The hydrogel was supplemented either with polyphosphate (polyP), administered as polyP•Ca2+-complex, or silica, or as biosilica that had been enzymatically prepared from ortho-silicate by silicatein. These hydrogels, together with SaOS-2 cells, were bioprinted to computer-designed scaffolds. The results revealed that bioglass (nano)particles, with a size of 55 nm and a molar ratio of SiO2∶CaO∶P2O5 of 55∶40∶5, did not affect the growth of the encapsulated cells. If silica, biosilica, or polyP•Ca2+-complex is co-added to the cell-containing alginate/gelatin hydrogel the growth behavior of the cells is not changed. Addition of 5 mg/ml of bioglass particles to this hydrogel significantly enhanced the potency of the entrapped SaOS-2 cells to mineralize. If compared with the extent of the cells to form mineral deposits in the absence of bioglass, the cells exposed to bioglass together with 100 µmoles/L polyP•Ca2+-complex increased their mineralization activity from 2.1- to 3.9-fold, or with 50 µmoles/L silica from 1.8- to 2.9-fold, or with 50 µmoles/L biosilica from 2.7- to 4.8-fold or with the two components together (100 µmoles/L polyP•Ca2+-complex and 50 µmoles/L biosilica) from 4.1- to 6.8-fold. Element analysis by EDX spectrometry of the mineral nodules formed by SaOS-2 revealed an accumulation of O, P, Ca and C, indicating that the mineral deposits contain, besides Ca-phosphate also Ca-carbonate. The results show that bioglass added to alginate/gelatin hydrogel increases the proliferation and mineralization of bioprinted SaOS-2 cells. We conclude that the development of cell-containing scaffolds consisting of a bioprintable, solid and cell-compatible inner matrix surrounded by a printable hard and flexible outer matrix containing bioglass, provide a suitable strategy for the fabrication of morphogenetically active and biodegradable implants.

Metastable amorphous calcium carbonate (ACC), formed as a precursor of crystalline carbonated apatite/hydroxyapatite in human bone, can be stabilized by inorganic polyphosphate. The resulting CaCO3/polyP, containing small amounts of vaterite, has osteogenic potential and potential application as a scaffold material for bone implants.