Biomineralization of Polyelectrolyte-Functionalized Electrospun Fibers: Optimization and In Vitro Validation for Bone Applications.

In recent years, polyelectrolytes have been successfully used as an alternative to non-collagenous proteins to promote interfibrillar biomineralization, to reproduce the spatial intercalation of mineral phases among collagen fibrils, and to design bioinspired scaffolds for hard tissue regeneration. Herein, hybrid nanofibers were fabricated via electrospinning, by using a mixture of Poly ɛ-caprolactone (PCL) and cationic cellulose derivatives, i.e., cellulose-bearing imidazolium tosylate (CIMD). The obtained fibers were self-assembled with Sodium Alginate (SA) by polyelectrolyte interactions with CIMD onto the fiber surface and, then, treated with simulated body fluid (SBF) to promote the precipitation of calcium phosphate (CaP) deposits. FTIR analysis confirmed the presence of SA and CaP, while SEM equipped with EDX analysis mapped the calcium phosphate constituent elements, estimating an average Ca/P ratio of about 1.33-falling in the range of biological apatites. Moreover, in vitro studies have confirmed the good response of mesenchymal cells (hMSCs) on biomineralized samples, since day 3, with a significant improvement in the presence of SA, due to the interaction of SA with CaP deposits. More interestingly, after a decay of metabolic activity on day 7, a relevant increase in cell proliferation can be recognized, in agreement with the beginning of the differentiation phase, confirmed by ALP results. Antibacterial tests performed by using different bacteria populations confirmed that nanofibers with an SA-CIMD complex show an optimal inhibitory response against S. mutans, S. aureus, and E. coli, with no significant decay due to the effect of CaP, in comparison with non-biomineralized controls. All these data suggest a promising use of these biomineralized fibers as bioinspired membranes with efficient antimicrobial and osteoconductive cues suitable to support bone healing/regeneration.

Development of collagen-chitosan dressing gel functionalized with propolis-zinc oxide nanoarchitectonics to accelerate wound healing.

Using an in situ sol-gel technique, new nanoarchitectonics of propolis loaded zinc oxide nanoarchitectonics (PP/ZnO-NPs) were developed in order to improve the in vivo outcomes of collagen-chitosan gel in wounded rats. The obtained nanoarchitectonics were fully characterized. The XRD results indicate the presence of a Zincite phase for ZnO-NPs and Zincite accompanied by a minor amount of zinc hydroxide for PP/ZnO-NPs samples. While the TEM findings illustrate the transfer of the ZnO-NPs from agglomerated spheres with an average particle size of 230 ± 29 nm to needle-like NPs of 323 ± 173 nm length (PP1/ZnO-NPs) and to a sheet-like NPs of 500 ± 173 nm diameter (PP2/ZnO-NPs). In addition, the incorporation of PP results in an increase in the surface negativity of ZnO-NPs to -31.4 ± 6.4 mV for PP2/ZnO-NPs. The antimicrobial activities of the nanocomposite gel loaded with 10%PP1/ZnO-NPs (G6) revealed the highest inhibition zone against E. coli (26 ± 2.31 mm). Remarkably, the in vivo outcomes showed that the nanocomposite gel (G6) has exceptional collagen deposition, quick wound closure rates, and re-epithelization. The outcomes demonstrate the nanocomposite gel encouraging biological properties for the treatment of damaged and infected wounds.

Cell migration, DNA fragmentation and antibacterial properties of novel silver doped calcium polyphosphate nanoparticles.

To combat infections, silver was used extensively in biomedical field but there was a need for a capping agent to eliminate its cytotoxic effects. In this study, polymeric calcium polyphosphate was doped by silver with three concentrations 1, 3 or 5 mol.% and were characterized by TEM, XRD, FTIR, TGA. Moreover, cytotoxicity, antibacterial, cell migration and DNA fragmentation assays were done to assure its safety. The results showed that the increase in silver percentage caused an increase in particle size. XRD showed the silver peaks, which indicated that it is present in its metallic form. The TGA showed that thermal stability was increased by increasing silver content. The antibacterial tests showed that the prepared nanoparticles have an antibacterial activity against tested pathogens. In addition, the cytotoxicity results showed that the samples exhibited non-cytotoxic behavior even with the highest doping concentration (5% Ag-CaPp). The cell migration assay showed that the increase in the silver concentration enhances cell migration up to 3% Ag-CaPp. The DNA fragmentation test revealed that all the prepared nanoparticles caused no fragmentation. From the results we can deduce that 3% Ag-CaPp was the optimum silver doped calcium polyphosphate concentration that could be used safely for medical applications.

Sodium Alginate- and Cationic Cellulose-Functionalized Polycaprolactone Nanofibers for In Vitro and Antibacterial Applications.

The use of polyelectrolytes is emerging as a fascinating strategy for the functionalization of biomedical membranes, due to their ability to enhance biological responses using the interaction effect of charged groups on multiple interface properties. Herein, two different polyelectrolytes were used to improve the antibacterial properties of polycaprolactone (PCL) nanofibers fabricated via electrospinning. First, a new cationic cellulose derivative, cellulose-bearing imidazolium tosylate (CIMD), was prepared via the nucleophilic substitution of the tosyl group using 1-methylimidazole, as confirmed by NMR analyses, and loaded into the PCL nanofibers. Secondly, sodium alginate (SA) was used to uniformly coat the fibers' surface via self-assembly, as remarked through SEM-EDX analyses. Polyelectrolyte interactions between the CIMD and the SA, initially detected using a FTIR analysis, were confirmed via Z potential measurements: the formation of a CMID/SA complex promoted a substantial charge neutralization of the fibers' surfaces with effects on the physical properties of the membrane in terms of water adsorption and in vitro degradation. Moreover, the presence of SA contributed to the in vitro response of human mesenchymal stem cells (hMSCs), as confirmed by a significant increase in the cells' viability after 7 days in the case of the PCL/CMID/SA complex with respect to the PCL and PCL/CMID membranes. Contrariwise, SA did not nullify the antibacterial effect of CMID, as confirmed by the comparable resistance exhibited by S. mutans, S. aureus, and E. coli to the PCL/CIMD and PCL/CIMD/SA membranes. All the reported results corroborate the idea that the CIMD/SA functionalization of PCL nanofibers has a great potential for the fabrication of efficient antimicrobial membranes for wound healing.

Egyptian propolis extract for functionalization of cellulose nanofiber/poly(vinyl alcohol) porous hydrogel along with characterization and biological applications.

Bee propolis is one of the most common natural extracts and has gained significant interest in biomedicine due to its high content of phenolic acids and flavonoids, which are responsible for the antioxidant activity of natural products. The present study report that the propolis extract (PE) was produced by ethanol in the surrounding environment. The obtained PE was added at different concentrations to cellulose nanofiber (CNF)/poly(vinyl alcohol) (PVA), and subjected to freezing thawing and freeze drying methods to develop porous bioactive matrices. Scanning electron microscope (SEM) observations displayed that the prepared samples had an interconnected porous structure with pore sizes in the range of 10-100 µm. The high performance liquid chromatography (HPLC) results of PE showed around 18 polyphenol compounds, with the highest amounts of hesperetin (183.7 µg/mL), chlorogenic acid (96.9 µg/mL) and caffeic acid (90.2 µg/mL). The antibacterial activity results indicated that both PE and PE-functionalized hydrogels exhibited a potential antimicrobial effects against Escherichia coli, Salmonella typhimurium, Streptococcus mutans, and Candida albicans. The in vitro test cell culture experiments indicated that the cells on the PE-functionalized hydrogels had the greatest viability, adhesion, and spreading of cells. Altogether, these data highlight the interesting effect of propolis bio-functionalization to enhance the biological features of CNF/PVA hydrogel as a functional matrix for biomedical applications.

Chitosan-organosilica hybrid decorated with silver nanoparticles for antimicrobial wearable cotton fabrics.

Functional cotton fabrics using silver-based nanoparticles (AgNPs) have attracted a lot of attention as a new generation of healthcare wearable textile. In this study, cotton fabrics were coated via impregnation with silver nanoparticles using chitosan (Cs) and (or) chitosan-organosilica (Cs-OSH) solutions as adhesives matrices. The physicochemical properties were studied using UV-VIS spectroscopy, and transmission electron microscopy (TEM) and scanning electron microscope coupled with energy-dispersive X-ray spectroscopy methods (SEM-EDX). The antibacterial activity of the silver-treated fabrics was determined using agar diffusion method. However, nanosize spherical AgNPs were observed in Cs and Cs-OSH solution. The average particle diameter was around 10 nm for Cs/AgNPs sample and close 21 nm for Cs-OSH/AgNPs. Microscopy images showed the deposition of Ag NPs on the surface of cotton fibers. The results indicated that the cotton fibers treated with Cs-OSH/AgNPs solution showed good stability against washing and maintained higher antimicrobial activity even after being exposed to 10 consecutive home laundering conditions. Thus, this work suggests the use of chitosan-organosilicon matrix to improve the bonding between AgNPs and cotton fibers for better and long-term antimicrobial activity.

A multicompartment vascular implant of electrospun wintergreen oil/ polycaprolactone fibers coated with poly(ethylene oxide).

BACKGROUND: The aim of the present study was to fabricate double layered scaffolds of electrospun polycaprolactone (PCL) and poly(ethylene oxide) (PEO). The electrospun PCL fibers were functionalized with wintergreen oil (WO) as a novel approach to prevent vascular grafts failure due to thrombosis by adjusting biomaterial-blood interactions. METHODS: PCL tubular scaffolds were prepared by electrospinning approach and coated with PEO as a hydrophilic polymer. The single and double layered scaffolds were characterized in terms of their morphological, chemical properties -as well as-hemocompatibility assays (i.e. prothrombin time, hemolysis percentage and platelets adhesion). Moreover, the antioxidant potential of WO-PCL samples were measured by 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH) free radical assay. RESULTS: The results demonstrated that incorporation of WO during the electrospinning process decreased the PCL fiber diameter. In addition, the prothrombine time assay shows that WO could be used to lower the electrospun PCL fiber tendency to induce blood clotting. Moreover, SEM observations of platelets adhesion of both single and double layered PCL/PEO scaffolds fiber shows an increase of platelets number, compared with the scaffolds containing WO. CONCLUSIONS: The antioxidant potential and blood compatibility measurements of WO-PCL/PEO samples highlight the approach made so far as an ideal synthetic small size vascular grafts to overcome autogenous grafts shortages and drawbacks.

Biomimetic Alginate/Gelatin Cross-Linked Hydrogels Supplemented with Polyphosphate for Wound Healing Applications.

In the present study, the fabrication of a biomimetic wound dressing that mimics the extracellular matrix, consisting of a hydrogel matrix composed of non-oxidized and periodate-oxidized marine alginate, was prepared to which gelatin was bound via Schiff base formation. Into this alginate/oxidized-alginate-gelatin hydrogel, polyP was stably but reversibly integrated by ionic cross-linking with Zn2+ ions. Thereby, a soft hybrid material is obtained, consisting of a more rigid alginate scaffold and porous structures formed by the oxidized-alginate-gelatin hydrogel with ionically cross-linked polyP. Two forms of the Zn-polyP-containing matrices were obtained based on the property of polyP to form, at neutral pH, a coacervate-the physiologically active form of the polymer. At alkaline conditions (pH 10), it will form nanoparticles, acting as a depot that is converted at pH 7 into the coacervate phase. Both polyP-containing hydrogels were biologically active and significantly enhanced cell growth/viability and attachment/spreading of human epidermal keratinocytes compared to control hydrogels without any adverse effect on reconstructed human epidermis samples in an in vitro skin irritation test system. From these data, we conclude that polyP-containing alginate/oxidized-alginate-gelatin hydrogels may provide a suitable regeneratively active matrix for wound healing for potential in vivo applications.

The biomaterial polyphosphate blocks stoichiometric binding of the SARS-CoV-2 S-protein to the cellular ACE2 receptor.

The effect of the polyanionic polymer of inorganic polyphosphate (polyP) involved in innate immunity on the binding of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein to the cellular ACE2 receptor was studied. The RBD surface comprises a basic amino acid stretch of four arginine residues which interact with the physiological polyP (polyP40) and polyP3. Subsequently, the interaction of RBD with ACE2 is sensitively inhibited. After the chemical modification of arginine, an increased inhibition by polyP, at a 1 : 1 molar ratio (polyP : RBP), is measured already at 0.1 µg mL-1. Heparin was ineffective. The results suggest a potential therapeutic benefit of polyP against SARS-CoV-2 infection.

Amplified morphogenetic and bone forming activity of amorphous versus crystalline calcium phosphate/polyphosphate.

Amorphous Ca-phosphate (ACP) particles stabilized by inorganic polyphosphate (polyP) were prepared by co-precipitation of calcium and phosphate in the presence of polyP (15% [w/w]). These hybrid nanoparticles showed no signs of crystallinity according to X-ray diffraction analysis, in contrast to the particles obtained at a lower (5% [w/w]) polyP concentration or to hydroxyapatite. The ACP/15% polyP particles proved to be a suitable matrix for cell growth and attachment and showed pronounced osteoblastic and vasculogenic activity in vitro. They strongly stimulated mineralization of the human osteosarcoma cell line SaOS-2, as well as cell migration/microvascularization, as demonstrated in the scratch assay and the in vitro angiogenesis tube forming assay. The possible involvement of an ATP gradient, generated by polyP during tube formation of human umbilical vein endothelial cells, was confirmed by ATP-depletion experiments. In order to assess the morphogenetic activity of the hybrid particles in vivo, experiments in rabbits using the calvarial bone defect model were performed. The particles were encapsulated in poly(d,l-lactide-co-glycolide) microspheres. In contrast, to crystalline Ca-phosphate (containing only 5% [w/w] polyP) or to crystalline ß-tricalcium phosphate, amorphous ACP/15% polyP particles caused pronounced osteoinductive activity already after a six-week healing period. The synthesis of new bone tissue was accompanied by an intense vascularization and an increased expression of mineralization/vascularization marker genes. The data show that amorphous polyP-stabilized ACP, which combines osteoinductive activity with the ability to act as a precursor of hydroxyapatite formation both in vitro and in vivo, is a promising material for bone regeneration.

Nanoparticle-directed and ionically forced polyphosphate coacervation: a versatile and reversible core-shell system for drug delivery.

A drug encapsulation/delivery system using a novel principle is described that is based on an intra-particle migration of calcium ions between a central Ca2+-enriched nanoparticle core and the surrounding shell compartment. The supply of Ca2+ is needed for the formation of a coacervate shell around the nanoparticles, acting as the core of drug-loadable core-shell particles, using the physiological inorganic polymer polyphosphate (polyP). This polyanion has the unique property to form, at an alkaline pH and in the presence of a stoichiometric surplus of calcium ions, water-insoluble and stabile amorphous nanoparticles. At neutral pH a coacervate, the biologically active form of the polymer, is obtained that is composed of polyP and Ca2+. The drug-loaded core-shell particles, built from the Ca-polyP core and the surrounding Ca-polyP shell, were fabricated in two successive steps. First, the formation of the nanoparticle core at pH 10 and a superstoichiometric 2:1 molar ratio between CaCl2 and Na-polyP into which dexamethasone, as a phosphate derivative, was incorporated. Second, the preparation of the coacervate shell, loaded with ascorbic acid, by exposure of the Ca-polyP core to soluble Na-polyP and L-ascorbate (calcium salt). EDX analysis revealed that during this step the Ca2+ ions required for coacervate formation migrate from the Ca-polyP core (with a high Ca:P ratio) to the shell. Electron microscopy of the particles show an electron-dense 150-200 nm sized core surrounded by a less sharply delimited electron-sparse shell. The core-shell particles exhibited strong osteogenic activity in vitro, based on the combined action of polyP and of dexamethasone and ascorbic acid, which reversibly bind to the anionic polyP via ionic Ca2+ bonds. Drug release from the particles occurs after contact with a peptide/protein-containing serum, a process which is almost complete after 10 days and accompanied by the conversion of the nanoparticles into a coacervate. Human osteosarcoma SaOS-2 cells cultivated onto or within an alginate hydrogel matrix showed increased growth/viability and mineralization when the hybrid particles containing dexamethasone and ascorbic acid were embedded in the matrix. The polyP-based core-shell particles have the potential to become a suitable, pH-responsive drug encapsulation/release system, especially for bone, cartilage and wound healing.

The inorganic polymer, polyphosphate, blocks binding of SARS-CoV-2 spike protein to ACE2 receptor at physiological concentrations.

Inorganic polyphosphate (polyP) is a morphogenetically active and metabolic energy-delivering physiological polymer that is released from blood platelets. Here, we show that polyP efficiently inhibits the binding of the envelope spike (S)-protein of the coronavirus SARS-CoV-2, the causative agent of COVID-19, to its host cell receptor ACE2 (angiotensin-converting enzyme 2). To stabilize polyP against the polyP-degrading alkaline phosphatase, the soluble polymer was encapsulated in silica/polyP nanoparticles. Applying a binding assay, soluble Na-polyP (sizes of 40 Pi and of 3 Pi units) as well as silica-nanoparticle-associated polyP significantly inhibit the interaction of the S-protein with ACE2 at a concentration of 1 µg/mL, close to the level present in blood. This inhibition is attributed to an interaction of polyP with a basic amino acid stretch on the surface of the receptor binding domain of S-protein. PolyP retains its activity in a flushing solution, opening a new strategy for the prevention and treatment of SARS-CoV-2 infection in the oropharyngeal cavity. The data suggest that supplementation of polyP might contribute to a strengthening of the human innate immunity system in compromised, thrombocytopenic COVID-19 patients.

A physiologically active interpenetrating collagen network that supports growth and migration of epidermal keratinocytes: zinc-polyP nanoparticles integrated into compressed collagen.

The distinguished property of the physiological polymer, inorganic polyphosphate (polyP), is to act as a bio-intelligent material which releases stimulus-dependent metabolic energy to accelerate wound healing. This characteristic is based on the bio-imitating feature of polyP to be converted, upon exposure to peptide-containing body fluids, from stable amorphous nanoparticles to a physiologically active and energy-delivering coacervate phase. This property of polyP has been utilized to fabricate a wound mat consisting of compressed collagen supplemented with amorphous polyP particles, formed from the inorganic polyanion with an over-stoichiometric ratio of zinc ions. The proliferation and the migration of human skin keratinocytes in those matrices were investigated. If the cells were embedded into the mat they respond with a significantly higher motility when zinc-polyP particles are present. Interestingly, only keratinocytes that were grown in a polyP environment developed well-structured microvilli, reflecting an increased biological activity. The data show that Zn-polyP particles incorporated into wound mats are a potent cell growth and cell migration-stimulating inorganic bio-material.

Amorphous Polyphosphate and Ca-Carbonate Nanoparticles Improve the Self-Healing Properties of both Technical and Medical Cements.

Cement is used both as a construction material and for medical applications. Previously, it has been shown that the physiological polymer inorganic polyphosphate (polyP) is morphogenetically active in regeneration of skin, bone, and cartilage. The present study investigates the question if this polymer is also a suitable additive to improve the self-healing capacity not only of construction cement but also of inorganic bone void fillers. For the application in the cement, two different polyP-based amorphous nanoparticles (NP) are prepared, amorphous Ca-polyP NP and amorphous Ca-carbonate (ACC) NP. The particles are integrated into poly(methyl methacrylate) in a concentration ratio of 1:10. This material applied onto Portland cement blocks either by brush application or by blow spinning strongly accelerates the self-healing property of the cement after a 10 day incubation period. Most likely, this process depends on bacteria and their membrane-associated alkaline phosphatase, resulting in the formation of calcite from ACC. In a second approach, polyP is integrated into a calcium-silicate-based cement used in reconstitutive medicine. Subsequently, the cement becomes softer and more elastic. The data show that bioinspired polyP/ACC NP are suitable additives to improve the self-healing of construction cement and to biologize bone cement.

Self-Healing Properties of Bioinspired Amorphous CaCO3/Polyphosphate-Supplemented Cement.

There is a strong interest in cement additives that are able to prevent or mitigate the adverse effects of cracks in concrete that cause corrosion of the reinforcement. Inorganic polyphosphate (polyP), a natural polymer that is synthesized by bacteria, even those on cement/concrete, can increase the resistance of concrete to progressive damage from micro-cracking. Here we use a novel bioinspired strategy based on polyP-stabilized amorphous calcium carbonate (ACC) to give this material self-healing properties. Portland cement was supplemented with ACC nanoparticles which were stabilized with 10% (w/w) Na-polyP. Embedding these particles in the hydrated cement resulted in the formation of calcite crystals after a hardening time of 10 days, which were not seen in controls, indicating that the particles dissolve and then transform into calcite. While there was no significant repair in the controls without ACC, almost complete closure of the cracks was observed after a 10 days healing period in the ACC-supplemented samples. Nanoindentation measurements on the self-healed crack surfaces showed a similar or slightly higher elasticity at a lower hardness compared to non-cracked surfaces. Our results demonstrate that bioinspired approaches, like the use of polyP-stabilized ACC shown here, can significantly improve the repair capacity of Portland cement.

Transformation of Construction Cement to a Self-Healing Hybrid Binder.

A new biomimetic strategy to im prove the self-healing properties of Portland cement is presented that is based on the application of the biogenic inorganic polymer polyphosphate (polyP), which is used as a cement admixture. The data show that synthetic linear polyp, with an average chain length of 40, as well as natural long-chain polyP isolated from soil bacteria, has the ability to support self-healing of this construction material. Furthermore, polyP, used as a water-soluble Na-salt, is subject to Na+/Ca2+ exchange by the Ca2+ from the cement, resulting in the formation of a water-rich coacervate when added to the cement surface, especially to the surface of bacteria-containing cement/concrete samples. The addition of polyP in low concentrations (<1% on weight basis for the solids) not only accelerated the hardening of cement/concrete but also the healing of microcracks present in the material. The results suggest that long-chain polyP is a promising additive that increases the self-healing capacity of cement by mimicking a bacteria-mediated natural mechanism.

In Situ Polyphosphate Nanoparticle Formation in Hybrid Poly(vinyl alcohol)/Karaya Gum Hydrogels: A Porous Scaffold Inducing Infiltration of Mesenchymal Stem Cells.

The preparation and characterization of a porous hybrid cryogel based on the two organic polymers, poly(vinyl alcohol) (PVA) and karaya gum (KG), into which polyphosphate (polyP) nanoparticles have been incorporated, are described. The PVA/KG cryogel is prepared by intermolecular cross-linking of PVA via freeze-thawing and Ca2+-mediated ionic gelation of KG to form stable salt bridges. The incorporation of polyP as amorphous nanoparticles with Ca2+ ions (Ca-polyP-NP) is achieved using an in situ approach. The polyP constituent does not significantly affect the viscoelastic properties of the PVA/KG cryogel that are comparable to natural soft tissue. The exposure of the Ca-polyP-NP within the cryogel to medium/serum allows the formation of a biologically active polyP coacervate/protein matrix that stimulates the growth of human mesenchymal stem cells in vitro and provides the cells a suitable matrix for infiltration superior to the polyP-free cryogel. In vivo biocompatibility studies in rats reveal that already two to four weeks after implantation into muscle, the implant regions containing the polyP-KG/PVA material become replaced by initial granulation tissue, whereas the controls are free of any cells. It is proposed that the polyP-KG/PVA cryogel has the potential to become a promising implant material for soft tissue engineering/repair.

Biomimetic transformation of polyphosphate microparticles during restoration of damaged teeth.

OBJECTIVE: In the present study, we investigated the fusion process between amorphous microparticles of the calcium salt of the physiological polymer comprising orthophosphate units, of inorganic polyphosphate (polyP), and enamel. METHODS: This polymer was incorporated as an ingredient into toothpaste and the fusion process was studied by electron microscopy and by synchrotron-based X-ray tomography microscopy (SRXTM) techniques. RESULTS: The data showed that toothpaste, supplemented with the amorphous Ca-polyP microparticles (aCa-polyP-MP), not only reseals tooth defects on enamel, like carious lesions, and dentin, including exposed dentinal tubules, but also has the potential to induce re-mineralization in the enamel and dentin regions. The formation of a regeneration mineralic zone on the tooth surface induced by aCa-polyP-MP was enhanced upon exposure to artificial saliva, as demonstrated by SRXTM. Energy dispersive X-ray analysis revealed an increase in the calcium/phosphorus atomic ratio of the enamel deposits to values characteristic for the particles during the treatment with polyP applied in the toothpaste, indicating a fusion of the particles with the tooth mineral. SIGNIFICANCE: Our results suggest that toothpaste enriched with aCa-polyP-MP is a promising biomimetic material for accelerating enamel and dentin restoration.

Role of ATP during the initiation of microvascularization: acceleration of an autocrine sensing mechanism facilitating chemotaxis by inorganic polyphosphate.

The in vitro tube formation assay with human umbilical vein endothelial cells (HUVEC) was applied to identify the extra- and intracellular sources of metabolic energy/ATP required for cell migration during the initial stage of microvascularization. Extracellularly, the physiological energy-rich polymer, inorganic polyphosphate (polyP), applied as biomimetic amorphous calcium polyP microparticles (Ca-polyP-MP), is functioning as a substrate for ATP generation most likely via the combined action of the alkaline phosphatase (ALP) and the adenylate kinase (AK). The linear Ca-polyP-MP with a size of 40 phosphate units, close to the polyP in the acidocalcisomes in the blood platelets, were found to increase endothelial cell tube formation, as well as the intracellular ATP levels. Depletion of extracellular ATP with apyrase suppressed tube formation during the initial incubation period. Inhibition experiments revealed that inhibitors (levamisole and Ap5A) of the enzymes involved in extracellular ATP generation strongly reduce the Ca-polyP-MP-induced tube formation. The stimulatory effect of Ca-polyP-MP was also diminished by the glycolysis inhibitor oxamate and trifluoperazine which blocks endocytosis, as well as by MRS2211, an antagonist of the P2Y13 receptor. Oligomycin, an inhibitor of the mitochondrial F0F1-ATP synthase, displayed no effect at lower concentrations on tube formation. Electron microscopic data revealed that after cellular uptake, the Ca-polyP-MP accumulate close to the cell membrane. We conclude that in HUVEC exposed to polyP, ATP is formed extracellularly via the coupled ALP-AK reaction, and intracellularly during glycolysis. The results suggest an autocrine signaling pathway of ATP with polyP as an extracellular store of metabolic energy for endothelial cell migration during the initial vascularization process.

Transformation of Amorphous Polyphosphate Nanoparticles into Coacervate Complexes: An Approach for the Encapsulation of Mesenchymal Stem Cells.

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 Ca2+ -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.