Energy level as a theranostic factor for successful therapy of tissue injuries with polyphosphate: the triad metabolic energy - mechanical energy - heat

Abstract

Rationale: Tissue regeneration of skin and bone is an energy-intensive, ATP-consuming process that, if impaired, can lead to the development of chronic clinical pictures. ATP levels in the extracellular space including the exudate of wounds, especially chronic wounds, are low. This deficiency can be compensated by inorganic polyphosphate (polyP) supplied via the blood platelets to the regenerating site. Methods: The contribution of the different forms of energy derived from polyP (metabolic energy, mechanical energy and heat) to regeneration processes was dissected and studied both in vitro and in patients. ATP is generated metabolically during the enzymatic cleavage of the energy-rich anhydride bonds between the phosphate units of polyP, involving the two enzymes alkaline phosphatase (ALP) and adenylate kinase (ADK). Exogenous polyP was administered after incorporation into compressed collagen or hydrogel wound coverages to evaluate its regenerative activity for chronic wound healing. Results: In a proof-of-concept study, fast healing of chronic wounds was achieved with the embedded polyP, supporting the crucial regeneration-promoting activity of ATP. In the presence of Ca²⁺ in the wound exudate, polyP undergoes a coacervation process leading to a conversion of fibroblasts into myofibroblasts, a crucial step supporting cell migration during regenerative tissue repair. During coacervation, a switch from an endothermic to an exothermic, heat-generating process occurs, reflecting a shift from an entropically- to an enthalpically-driven thermodynamic reaction. In addition, mechanical forces cause the appearance of turbulent flows and vortices during liquid-liquid phase separation. These mechanical forces orient the cellular and mineralic (hydroxyapatite crystallite) components, as shown using mineralizing SaOS-2 cells as a model. Conclusion: Here we introduce the energetic triad: metabolic energy (ATP), thermal energy and mechanical energy as a novel theranostic biomarker, which contributes essentially to a successful application of polyP for regeneration processes.

Keywords: Energy conversion; Metabolic energy; Polyphosphate; Thermodynamics; Wound healing.

Figure 1

ATP level in wound exudate. As a control, an aliquot of wound exudate from healthy individuals was centrifuged prior to testing in the luminescence assay after supplementing with 10 µg/mL Na-polyP; the resulting sediment was suspended in saline and tested. Similarly, the aliquots of wound exudate from chronic wounds and from healthy individuals were tested (n=6). Means (in pmol/mL) ± SD are given; the significance is indicated (* P < 0.01).

Figure 2

Fabrication of the two wound closures applied to human chronic wounds: (I.) (A) Collagen-based mat. Collagen, was pretreated by a pH shift (pH 3 to pH 7) to allow (B and C) a lateral alignment of the collagen bundles. The 1 mm thick mats (shown in A) were supplemented with polyP (5 mg of Ca-polyP-NP per g of wet collagen mat). (D) The integrated NP (np) measured ≈100 nm. Separately, a wound gel (E to H) based on hydroxyethyl cellulose hydrogel (hg) was prepared with 600 μg/mL of Na-polyP and 60 μg/mL of Ca-polyP-NP. After transfer of the hydrogel into the wound exudate, the coacervate (coa) formation started, (G) a process during which the cells (c) within the exudate assemble in the polyP-rich (polyP) layers, which are separated from the aqueous layer (aq). (II.) Application of the two polyP wound coverages for the healing of chronic wounds in a human patient. (A) The initial deeply infiltrating basal-cell carcinoma tumor was extensively excised (B). Since the ≈80 mm large wound (C) did not heal (the cranial bone was still open), (D) a polyP-enriched collagen mat layered into the wound and every three days the lesion was moistened with the polyP-containing wetting solution. Now, rapid healing occurred and granulation tissue developed. Then, (E) treatment with the polyP-hydrogel started. The healing progress continued and (F) allowed a covering with a split skin graft. After 6 weeks of polyP treatment the patient was released home.

Figure 3

Characterization of the cells within the collagen-based mats and biopsy samples. (I.) Cells from collagen (col) mats used for chronic wound patients. (A to C) In polyP-free medium/FCS, the cells remain rounded (c(r)). (D to F) In contrast, after transfer of the cells into polyP medium/FCS (50 µg/mL Na-polyP), their morphology changed to an elongated/fusiform (c(fu)) shape during the 12 h incubation period. (II.) Biopsies taken from the collagen-based mats. The biopsies were dissected into two regions, a region adjacent to the polyP-enriched mat and the distal part embedded in the granulation tissue. (A) Using urea polyacrylamide gel electrophoresis (PAGE), (A-a) only little polyP is present in the distal region, whereas (A-b) a strong and distinct signal for polyP with a size of ≈30 P_i units is found in the section neighboring the polyP-mat. (B) Tissue section through the biopsy with the terminal mat (ma) and the distal regenerating granulation tissue (gr-t). The regions from where the samples were taken for PAGE are marked with (a) and (b), corresponding to the PAGE lanes in (A). (C and D) Histology after staining with DRAQ5-positive nuclei in blue (c(n)) and strongly actin-positive cells (in red); these spindle-shaped cells (c(act+)) are fusiform myofibroblasts. (E and F) In contrast, the fibroblasts within the regenerating regions have a rounded shape and lack distinct actin protrusions (c(act-)). (G and H) Longitudinal section through the distal regenerating granulation tissue area, highlighting the size differences between the fibroblasts (c-fib), measuring 10-15 µm, and the larger myofibroblasts with >60 µm (c-myo-fib) that were specifically stained for actin with rhodamine/phalloidin.

Figure 4

The energetic triad. (I.) ATP generates from polyP through enzymatic processing of polyP by ALP and ADK. The energy, stored in the phosphoanhydride bond of ATP (ΔH_ATP) is split into energy used for heat production (Q), energy used for mechanical work (e.g., for cell migration) (W) and energy used for metabolic reactions (ΔH_P). These three forms of energy drive the three energy segments heat production, mechanical work, and biochemical bond formation. (II.) ATP the key driver for chronic wound healing. Initially, the available low extracellular ATP pool (Gibbs free energy, ΔG_ATP) is fueled into the energetic triad (heat energy, Q; energy for work, W; and metabolic energy used for metabolic reactions/product formation, ΔG_P). When the extracellular ATP pool depletes, the polyP metabolizing enzymes ALP and ADK refill the depleting ATP pool. The produced heat (Q) is used for accelerating the biochemical reactions according to the Van 't Hoff equation and keeps the thermodynamic system running.

Figure 5

Coacervation, a dynamic process. (I.) Addition of Ca²⁺ ions to Na-polyP causes the formation (A to C) of the coacervate through a liquid-liquid phase separation, in which initially a denser phase composed of polyP and a more dilute phase (mainly Ca²⁺ ions) are formed; optical microscopy. (D to F) Finally more stable Ca²⁺-polyP ribbons are assembled; SEM. (II.) A schematic outline of the three phases during coacervation, dissociation (endothermic reaction), turbulence and final assembly in dynamic layers (exothermic reaction).

Figure 6

Coacervation process. (I.) Thermographic profile (A) at 3.5°C during the initial dissociation phase of the reaction, Na-polyP and CaCl₂. During this endothermic phase, the temperature of the reaction mixture decreases (by 1.1°C). In this phase (A-a and A-b) no coacervates can be seen; only clusters of grains (gr) are visible. (B) The final phase of coacervation (at 37°C) is characterized by (B-a and B-b; optical microscopy) highly ordered coacervate blocks. This is an exothermic process and the temperature increases (by 0.7°C). (II.) PolyP coacervate formation in wound exudate. (A to C) PolyP-containing collagen mats are transferred to McCoy's medium/FCS and incubated for up to 36 h. During this period, coacervate (coa) clusters are rapidly formed; SEM. (D to F) Activation of cells in collagen mats from patients. Cells (c) residing in medium/FCS (with 50 µg/mL Na-polyP) change their morphology and turn from (D) spheroid to (E and F) elongated/fusiform, as seen after fluorescence staining with calcein AM.

Figure 7

HA mineralization is paralleled with metabolic energy consumption. (I.) Element distribution on SaOS-2 cells during HA mineralization in culture medium. Two days after seeding, the culture medium was enriched with the mineralization activation cocktail MAC. (A) After incubation for 5 days, the cell layers were inspected using Auriga FE-SEM microscopy to assess the element distribution. The nodules (no) consist of phosphorus and oxygen, while the background with cells (c) is highlighted in reddish. (B) Using the secondary electron image technique, the nodules appear in grey. (II.) Regional ATP distribution across the surface of the SaOS-2 cell layer. (A) Heavily mineralizing SaOS-2 cells with their protruding mineral nodule (no) deposits. (B) Regional ATP distribution, assessed semi-quantitatively. The higher levels of ATP/ATP colored in dark/blue match with the nodule formations, while the surrounding regions are bright red.

Figure 8

Influence of coacervation turbulence on the orientation pattern of the crystallites on SaOS-2 cells. (I.) Sketch of the experimental setup. (A) Cells were embedded in an alginate-based matrix, hardened with Ca²⁺ and enriched with Na-polyP (left panel); no contact of the cells is possible. (B) The matrix was prepared first without Na-polyP; then this polymer was added as an overlay to cells growing in medium/serum; coacervation was initiated by adding Ca²⁺. (C) Pattern of crystallites (crys) on SaOS-2 cells not exposed to coacervation. (D) Linearly arranged crystallites (crys) on the surfaces of cells within the coacervate vortices. (II.) Mineralizing nodules of cells within the alginate matrix and of cells on top of the matrix; SEM; ESEM. (A to F) The mineralizing nodules (no) of cells within the matrix, not exposed to the mechanical flow energy caused by coacervate formation, are arranged in a random pattern on the cell (c) layer (A to C) in the first phase at day 1 to day 3. As the mineral nodules grow, they fuse together (day 3 (d: 3) to day 4) and are (D to F) marginally separated by cell extensions. (G to L) The organizational pattern of the nodules is significantly different if the cells were directly exposed to the coacervate movements/fluxes. Initially, (G to I) the pattern of the nodules is again random (day 1 to day 3), while in the final phase (J to L) the cells change their morphology and the fusiform protrusions arrange the nodules in a threadlike pattern during the 4-day incubation period.