Polyphosphate Nanoparticles: Balancing Energy Requirements in Tissue Regeneration Processes.

Nanoparticles of a particular, evolutionarily old inorganic polymer found across the biological kingdoms have attracted increasing interest in recent years not only because of their crucial role in metabolism but also their potential medical applicability: it is inorganic polyphosphate (polyP). This ubiquitous linear polymer is composed of 10-1000 phosphate residues linked by high-energy anhydride bonds. PolyP causes induction of gene activity, provides phosphate for bone mineralization, and serves as an energy supplier through enzymatic cleavage of its acid anhydride bonds and subsequent ATP formation. The biomedical breakthrough of polyP came with the development of a successful fabrication process, in depot form, as Ca- or Mg-polyP nanoparticles, or as the directly effective polymer, as soluble Na-polyP, for regenerative repair and healing processes, especially in tissue areas with insufficient blood supply. Physiologically, the platelets are the main vehicles for polyP nanoparticles in the circulating blood. To be biomedically active, these particles undergo coacervation. This review provides an overview of the properties of polyP and polyP nanoparticles for applications in the regeneration and repair of bone, cartilage, and skin. In addition to studies on animal models, the first successful proof-of-concept studies on humans for the healing of chronic wounds are outlined.

Biomimetic Polyphosphate Materials: Toward Application in Regenerative Medicine.

In recent years, inorganic polyphosphate (polyP) has attracted increasing attention as a biomedical polymer or biomaterial with a great potential for application in regenerative medicine, in particular in the fields of tissue engineering and repair. The interest in polyP is based on two properties of this physiological polymer that make polyP stand out from other polymers: polyP has morphogenetic activity by inducing cell differentiation through specific gene expression, and it functions as an energy store and donor of metabolic energy, especially in the extracellular matrix or in the extracellular space. No other biopolymer applicable in tissue regeneration/repair is known that is endowed with this combination of properties. In addition, polyP can be fabricated both in the form of a biologically active coacervate and as biomimetic amorphous polyP nano/microparticles, which are stable and are activated by transformation into the coacervate phase after contact with protein/body fluids. PolyP can be used in the form of various metal salts and in combination with various hydrogel-forming polymers, whereby (even printable) hybrid materials with defined porosities and mechanical and biological properties can be produced, which can even be loaded with cells for 3D cell printing or with drugs and support the growth and differentiation of (stem) cells as well as cell migration/microvascularization. Potential applications in therapy of bone, cartilage and eye disorders/injuries and wound healing are summarized and possible mechanisms are discussed.

Polyphosphate in Chronic Wound Healing: Restoration of Impaired Metabolic Energy State.

Many pathological conditions are characterized by a deficiency of metabolic energy. A prominent example is nonhealing or difficult-to-heal chronic wounds. Because of their unique ability to serve as a source of metabolic energy, inorganic polyphosphates (polyP) offer the opportunity to develop novel strategies to treat such wounds. The basis is the generation of ATP from the polymer through the joint action of two extracellular or plasma membrane-bound enzymes alkaline phosphatase and adenylate kinase, which enable the transfer of energy-rich phosphate from polyP to AMP with the formation of ADP and finally ATP. Building on these findings, it was possible to develop novel regeneratively active materials for wound therapy, which have already been successfully evaluated in first studies on patients.

Caged Dexamethasone/Quercetin Nanoparticles, Formed of the Morphogenetic Active Inorganic Polyphosphate, are Strong Inducers of MUC5AC.

Inorganic polyphosphate (polyP) is a widely distributed polymer found from bacteria to animals, including marine species. This polymer exhibits morphogenetic as well as antiviral activity and releases metabolic energy after enzymatic hydrolysis also in human cells. In the pathogenesis of the coronavirus disease 2019 (COVID-19), the platelets are at the frontline of this syndrome. Platelets release a set of molecules, among them polyP. In addition, the production of airway mucus, the first line of body defense, is impaired in those patients. Therefore, in this study, amorphous nanoparticles of the magnesium salt of polyP (Mg-polyP-NP), matching the size of the coronavirus SARS-CoV-2, were prepared and loaded with the secondary plant metabolite quercetin or with dexamethasone to study their effects on the respiratory epithelium using human alveolar basal epithelial A549 cells as a model. The results revealed that both compounds embedded into the polyP nanoparticles significantly increased the steady-state-expression of the MUC5AC gene. This mucin species is the major mucus glycoprotein present in the secreted gel-forming mucus. The level of gene expression caused by quercetin or with dexamethasone, if caged into polyP NP, is significantly higher compared to the individual drugs alone. Both quercetin and dexamethasone did not impair the growth-supporting effect of polyP on A549 cells even at concentrations of quercetin which are cytotoxic for the cells. A possible mechanism of the effects of the two drugs together with polyP on mucin expression is proposed based on the scavenging of free oxygen species and the generation of ADP/ATP from the polyP, which is needed for the organization of the protective mucin-based mucus layer.

Morphogenetic (Mucin Expression) as Well as Potential Anti-Corona Viral Activity of the Marine Secondary Metabolite Polyphosphate on A549 Cells.

The mucus layer of the nasopharynx and bronchial epithelium has a barrier function against inhaled pathogens such as the coronavirus SARS-CoV-2. We recently found that inorganic polyphosphate (polyP), a physiological, metabolic energy (ATP)-providing polymer released from blood platelets, blocks the binding of the receptor binding domain (RBD) to the cellular ACE2 receptor in vitro. PolyP is a marine natural product and is abundantly present in marine bacteria. Now, we have approached the in vivo situation by studying the effect of polyP on the human alveolar basal epithelial A549 cells in a mucus-like mucin environment. These cells express mucins as well as the ectoenzymes alkaline phosphatase (ALP) and adenylate kinase (ADK), which are involved in the extracellular production of ATP from polyP. Mucin, integrated into a collagen-based hydrogel, stimulated cell growth and attachment. The addition of polyP to the hydrogel significantly increased cell attachment and also the expression of the membrane-tethered mucin MUC1 and the secreted mucin MUC5AC. The increased synthesis of MUC1 was also confirmed by immunostaining. This morphogenetic effect of polyP was associated with a rise in extracellular ATP level. We conclude that the nontoxic and non-immunogenic polymer polyP could possibly also exert a protective effect against SARS-CoV-2-cell attachment; first, by stimulating the innate antiviral response by strengthening the mucin barrier with its antimicrobial proteins, and second, by inhibiting virus attachment to the cells, as deduced from the reduction in the strength of binding between the viral RBD and the cellular ACE2 receptor.

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.

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.

Polyphosphate as a donor of high-energy phosphate for the synthesis of ADP and ATP.

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.

Inorganic polyphosphate induces accelerated tube formation of HUVEC endothelial cells.

In this study, the effect of inorganic polyphosphate (polyP) on the initial phase of angiogenesis and vascularization was investigated, applying the HUVEC cell tube formation assay. PolyP is a physiological and high energy phosphate polymer which has been proposed to act as a metabolic fuel in the extracellular space with only a comparably low ATP content. The experiments revealed that polyP accelerates tube formation of human umbilical vein endothelial cells (HUVEC), seeded onto a solidified basement membrane extract matrix which contains polyP-metabolizing alkaline phosphatase (ALP) activity. This effect is abolished by co-addition of apyrase, which degrades ATP to AMP and inorganic phosphate. The assumption that ATP, derived from polyP, activates HUVEC cells leading to tube formation was corroborated by experiments showing that addition of polyP to the cells causes a strong rise of ATP level in the culture medium. Finally, we show that at a later stage of cultivation of HUVEC cells, after 3 d, polyP causes a strong enhancement of the expression of the genes encoding for the two major matrix metalloproteinases (MMPs) released by endothelial cells during tube formation, MMP-9 and MMP-2. This stimulatory effect is again abrogated by addition of apyrase together with polyP. From these results, we propose that polyP is involved either directly or indirectly in energy supply, via ALP-mediated transfer of energy-rich phosphate under ATP formation. This ATP is utilized for the activation and oriented migration of endothelial cells and for the matrix organization during the initial phases of tube formation.

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.

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.

Efficient Carrier-to-Exciton Conversion in Field Emission Tunnel Diodes Based on MIS-Type van der Waals Heterostack.

We report on efficient carrier-to-exciton conversion and planar electroluminescence from tunnel diodes based on a metal-insulator-semiconductor (MIS) van der Waals heterostack consisting of few-layer graphene (FLG), hexagonal boron nitride (hBN), and monolayer tungsten disulfide (WS2). These devices exhibit excitonic electroluminescence with extremely low threshold current density of a few pA·µm-2, which is several orders of magnitude lower compared to the previously reported values for the best planar EL devices. Using a reference dye, we estimate the EL quantum efficiency to be ∼1% at low current density limit, which is of the same order of magnitude as photoluminescence quantum yield at the equivalent excitation rate. Our observations reveal that the efficiency of our devices is not limited by carrier-to-exciton conversion efficiency but by the inherent exciton-to-photon yield of the material. The device characteristics indicate that the light emission is triggered by injection of hot minority carriers (holes) to n-doped WS2 by Fowler-Nordheim tunneling and that hBN serves as an efficient hole-transport and electron-blocking layer. Our findings offer insight into the intelligent design of van der Waals heterostructures and avenues for realizing efficient excitonic devices.

Amorphous, Smart, and Bioinspired Polyphosphate Nano/Microparticles: A Biomaterial for Regeneration and Repair of Osteo-Articular Impairments In-Situ.

Using femur explants from mice as an in vitro model, we investigated the effect of the physiological polymer, inorganic polyphosphate (polyP), on differentiation of the cells of the bone marrow in their natural microenvironment into the osteogenic and chondrogenic lineages. In the form of amorphous Ca-polyP nano/microparticles, polyP retains its function to act as both an intra- and extracellular metabolic fuel and a stimulus eliciting morphogenetic signals. The method for synthesis of the nano/microparticles with the polyanionic polyP also allowed the fabrication of hybrid particles with the bisphosphonate zoledronic acid, a drug used in therapy of bone metastases in cancer patients. The results revealed that the amorphous Ca-polyP particles promote the growth/viability of mesenchymal stem cells, as well as the osteogenic and chondrogenic differentiation of the bone marrow cells in rat femur explants, as revealed by an upregulation of the expression of the transcription factors SOX9 (differentiation towards osteoblasts) and RUNX2 (chondrocyte differentiation). In parallel to this bone anabolic effect, incubation of the femur explants with these particles significantly reduced the expression of the gene encoding the osteoclast bone-catabolic enzyme, cathepsin-K, while the expression of the tartrate-resistant acid phosphatase remained unaffected. The gene expression data were supported by the finding of an increased mineralization of the cells in the femur explants in response to the Ca-polyP particles. Finally, we show that the hybrid particles of polyP complexed with zoledronic acid exhibit both the cytotoxic effect of the bisphosphonate and the morphogenetic and mineralization inducing activity of polyP. Our results suggest that the Ca-polyP nano/microparticles are not only a promising scaffold material for repairing long bone osteo-articular damages but can also be applied, as a hybrid with zoledronic acid, as a drug delivery system for treatment of bone metastases. The polyP particles are highlighted as genuine, smart, bioinspired nano/micro biomaterials.

Biocalcite and Carbonic Acid Activators.

Based on evolution of biomineralizing systems and energetic considerations, there is now compelling evidence that enzymes play a driving role in the formation of the inorganic skeletons from the simplest animals, the sponges, up to humans. Focusing on skeletons based on calcium minerals, the principle enzymes involved are the carbonic anhydrase (formation of the calcium carbonate-based skeletons of many invertebrates like the calcareous sponges, as well as deposition of the calcium carbonate bioseeds during human bone formation) and the alkaline phosphatase (providing the phosphate for bone calcium phosphate-hydroxyapatite formation). These two enzymes, both being involved in human bone formation, open novel not yet exploited targets for pharmacological intervention of human bone diseases like osteoporosis, using compounds that act as activators of these enzymes. This chapter focuses on carbonic anhydrases of biomedical interest and the search for potential activators of these enzymes, was well as the interplay between carbonic anhydrase-mediated calcium carbonate bioseed synthesis and metabolism of energy-rich inorganic polyphosphates. Beyond that, the combination of the two metabolic products, calcium carbonate and calcium-polyphosphate, if applied in an amorphous form, turned out to provide the basis for a new generation of scaffold materials for bone tissue engineering and repair that are, for the first time, morphogenetically active.

Morphogenetically-Active Barrier Membrane for Guided Bone Regeneration, Based on Amorphous Polyphosphate.

We describe a novel regeneratively-active barrier membrane which consists of a durable electrospun poly(ε-caprolactone) (PCL) net covered with a morphogenetically-active biohybrid material composed of collagen and inorganic polyphosphate (polyP). The patch-like fibrous collagen structures are decorated with small amorphous polyP nanoparticles (50 nm) formed by precipitation of this energy-rich and enzyme-degradable (alkaline phosphatase) polymer in the presence of calcium ions. The fabricated PCL-polyP/collagen hybrid mats are characterized by advantageous biomechanical properties, such as enhanced flexibility and stretchability with almost unaltered tensile strength of the PCL net. The polyP/collagen material promotes the attachment and increases the viability/metabolic activity of human mesenchymal stem cells compared to cells grown on non-coated mats. The gene expression studies revealed that cells, growing onto polyP/collagen coated mats show a significantly (two-fold) higher upregulation of the steady-state-expression of the angiopoietin-2 gene used as an early marker for wound healing than cells cultivated onto non-coated mats. Based on our results we propose that amorphous polyP, stabilized onto a collagen matrix, might be a promising component of functionally-active barrier membranes for guided tissue regeneration in medicine and dentistry.

Rebalancing ß-Amyloid-Induced Decrease of ATP Level by Amorphous Nano/Micro Polyphosphate: Suppression of the Neurotoxic Effect of Amyloid ß-Protein Fragment 25-35.

Morbus Alzheimer neuropathology is characterized by an impaired energy homeostasis of brain tissue. We present an approach towards a potential therapy of Alzheimer disease based on the high-energy polymer inorganic polyphosphate (polyP), which physiologically occurs both in the extracellular and in the intracellular space. Rat pheochromocytoma (PC) 12 cells, as well as rat primary cortical neurons were exposed to the Alzheimer peptide Aß25-35. They were incubated in vitro with polyphosphate (polyP); ortho-phosphate was used as a control. The polymer remained as Na⁺ salt; or complexed in a stoichiometric ratio to Ca2+ (Na-polyP[Ca2+]); or was processed as amorphous Ca-polyP microparticles (Ca-polyP-MP). Ortho-phosphate was fabricated as crystalline Ca-phosphate nanoparticles (Ca-phosphate-NP). We show that the pre-incubation of PC12 cells and primary cortical neurons with polyP protects the cells against the neurotoxic effect of the Alzheimer peptide Aß25-35. The strongest effect was observed with amorphous polyP microparticles (Ca-polyP-MP). The effect of the soluble sodium salt; Na-polyP (Na-polyP[Ca2+]) was lower; while crystalline orthophosphate nanoparticles (Ca-phosphate-NP) were ineffective. Ca-polyP-MP microparticles and Na-polyP[Ca2+] were found to markedly enhance the intracellular ATP level. Pre-incubation of Aß25-35 during aggregate formation, with the polyP preparation before exposure of the cells, had a small effect on neurotoxicity. We conclude that recovery of the compromised energy status in neuronal cells by administration of nontoxic biodegradable Ca-salts of polyP reverse the ß-amyloid-induced decrease of adenosine triphosphate (ATP) level. This study contributes to a new routes for a potential therapeutic intervention in Alzheimer's disease pathophysiology.

Effect of oxygen and ozone on p-type doping of ultra-thin WSe2 and MoSe2 field effect transistors.

We report on the p-type doping effect of oxygen and ozone molecules on mono- and few-layer WSe2 and MoSe2 field effect transistors. We show that adsorption of oxygen and ozone under ambient conditions results in subtantial doping and corresponding enhancement in the hole conductivity of the devices. Ozone-induced doping is found to be rapid and efficient, saturating within minutes of exposure whereas oxygen-induced doping occurs over a period of days to reach the equivalent level of doping. Our observations reveal that the water adlayer on the material surface plays a crucial role in solubilizing oxygen and ozone and in forming a redox couple with a large chemical potential.

Transport properties of monolayer MoS2 grown by chemical vapor deposition.

Recent success in the growth of monolayer MoS2 via chemical vapor deposition (CVD) has opened up prospects for the implementation of these materials into thin film electronic and optoelectronic devices. Here, we investigate the electronic transport properties of individual crystallites of high quality CVD-grown monolayer MoS2. The devices show low temperature mobilities up to 500 cm(2) V(-1) s(-1) and a clear signature of metallic conduction at high doping densities. These characteristics are comparable to the electronic properties of the best mechanically exfoliated monolayers in literature, verifying the high electronic quality of the CVD-grown materials. We analyze the different scattering mechanisms and show that the short-range scattering plays a dominant role in the highly conducting regime at low temperatures. Additionally, the influence of optical phonons as a limiting factor is discussed.

The Physiological Inorganic Polymers Biosilica and Polyphosphate as Key Drivers for Biomedical Materials in Regenerative Nanomedicine.

There is a need for novel nanomaterials with properties not yet exploited in regenerative nanomedicine. Based on lessons learned from the oldest metazoan phylum, sponges, it has been recognized that two previously ignored or insufficiently recognized principles play an essential role in tissue regeneration, including biomineral formation/repair and wound healing. Firstly, the dependence on enzymes as a driving force and secondly, the availability of metabolic energy. The discovery of enzymatic synthesis and regenerative activity of amorphous biosilica that builds the mineral skeleton of siliceous sponges formed the basis for the development of successful strategies for the treatment of osteochondral impairments in humans. In addition, the elucidation of the functional significance of a second regeneratively active inorganic material, namely inorganic polyphosphate (polyP) and its amorphous nanoparticles, present from sponges to humans, has pushed forward the development of innovative materials for both soft (skin, cartilage) and hard tissue (bone) repair. This energy-rich molecule exhibits a property not shown by any other biopolymer: the delivery of metabolic energy, even extracellularly, necessary for the ATP-dependent tissue regeneration. This review summarizes the latest developments in nanobiomaterials based on these two evolutionarily old, regeneratively active materials, amorphous silica and amorphous polyP, highlighting their specific, partly unique properties and mode of action, and discussing their possible applications in human therapy. The results of initial proof-of-concept studies on patients demonstrating complete healing of chronic wounds are outlined.

Acceleration of Wound Healing through Amorphous Calcium Carbonate, Stabilized with High-Energy Polyphosphate.

Amorphous calcium carbonate (ACC), precipitated in the presence of inorganic polyphosphate (polyP), has shown promise as a material for bone regeneration due to its morphogenetic and metabolic energy (ATP)-delivering properties. The latter activity of the polyP-stabilized ACC ("ACC∙PP") particles is associated with the enzymatic degradation of polyP, resulting in the transformation of ACC into crystalline polymorphs. In a novel approach, stimulated by these results, it was examined whether "ACC∙PP" also promotes the healing of skin injuries, especially chronic wounds. In in vitro experiments, "ACC∙PP" significantly stimulated the migration of endothelial cells, both in tube formation and scratch assays (by 2- to 3-fold). Support came from ex vivo experiments showing increased cell outgrowth in human skin explants. The transformation of ACC into insoluble calcite was suppressed by protein/serum being present in wound fluid. The results were confirmed in vivo in studies on normal (C57BL/6) and diabetic (db/db) mice. Topical administration of "ACC∙PP" significantly accelerated the rate of re-epithelialization, particularly in delayed healing wounds in diabetic mice (day 7: 1.5-fold; and day 13: 1.9-fold), in parallel with increased formation/maturation of granulation tissue. The results suggest that administration of "ACC∙PP" opens a new strategy to improve ATP-dependent wound healing, particularly in chronic wounds.