METHODS AND COMPOSITIONS FOR PREVENTION AND TREATMENT OF PRESSURE ULCERS

Abstract

The technology subject of the present application aims at providing a methodology for prevention of pressure ulcers.

Claims

1. A method for preventing pressure ulcers or injuries in a subject, the method comprising administering at least one pyruvate compound to a subject to thereby treat microscopic damage to a skin region or tissue region due to an intense or prolonged pressure or pressure in combination with shear, wherein the method excludes treatment of existing or visible pressure injuries.

2. The method according to claim 1, wherein said compound is pyruvic acid or an ester, amide or salt thereof.

3. The method according to claim 1, wherein said compound is pyruvic acid.

4. (canceled)

5. The method according to claim 42, wherein the ester is selected from alkyl esters and substituted forms thereof, wherein the alkyl ester is optionally derived from alcohols having between 1 and 10 carbon atoms.

6-7. (canceled)

8. The method according to claim 4, wherein the pyruvate ester is methyl pyruvate, ethyl pyruvate or propyl pyruvate.

9. (canceled)

10. The method according to claim 2, wherein the amide is derived from an amine selected from ammonia, mono-alkyl amine, di-alkyl amine and tri-alkyl amine, wherein the alkyl comprises from 1 to 10 carbon atoms.

11. The method according to claim 2, wherein said compound is a salt of pyruvic acid.

12. The method according to claim 11, wherein the salt is a base-addition salt derived from an organic or inorganic base.

13. The method according to claim 11, wherein the salt is selected from sodium pyruvate, calcium pyruvate, zinc pyruvate, lithium pyruvate, potassium pyruvate, magnesium pyruvate and manganese pyruvate.

14. The method according to claim 13, wherein the salt is sodium pyruvate.

15-29. (canceled)

30. The method according to claim 1, wherein the at least one pyruvate compound is administered with a low- to medium-level of a stretching force or deformation or strain to the skin region or to the deeper tissue region or to the organ of the subject, wherein said administration is prior to, subsequently to, concomitantly with or after administration of the pyruvate compound.

31-48. (canceled)

49. A device or an article comprising a releasable pyruvate compound, the device or article being configured and operable for prevention of pressure ulcers, and selected from a dressing, a pad, a bandage, an underwear, a diaper, a plaster, a patch, a mattress, a cushion, a seating surface, a surgical table, an examination table, a continuous positive airway pressure mask, an oxygen mask, a spinal board, a cervical collar, a pulse oximeter, a catheter, wiring, an electrode, a tracheostomy device, a nasogastric tube, an endotracheal tube, compression stockings, a cast, a positioner, a heel boot, a headrest, a footrest, a lying surface and a cover.

50. A device for inducing deformation of a skin region of a subject, the device comprising: an outer, non-deformable or minimally-deformable frame element, having at least one first skin-contacting surface configured to adhere to a first skin region of the subject; at least one inner, elastically, visco-elastically or plastically deformable element, having at least one second skin-contacting surface that is configured to adhere to a second skin region of the subject; wherein the second skin region is within boundaries formed by the outer frame element; and a member disposed between and connecting said outer element and inner element; wherein the member is elastic, viscoelastic or plastic; wherein the elastic, viscoelastic or plastic member is configured to be selectively deformable, thereby causing elastic, viscoelastic or plastic deformation of the at least one inner element and said second skin region.

51-52. (canceled)

53. The device according to claim 50, wherein the inner frame is concentric with the outer frame such that the inner frame is in contact with a skin region that is contained within boundaries formed by the outer ring.

54. The device according to claim 50, wherein the elastic, viscoelastic of plastic member is configured to selectively deform upon application of at least one of a mechanical force, a magnetic force, a magneto-mechanical force, an electrical input, a chemical reaction, a temperature change and at least one skin-environmental condition or any combination thereof.

55. The device according to claim 54, wherein the elastic, viscoelastic, or plastic member is induced to deform in response to at least one skin-environmental condition selected from exposure to air, skin, body-heat, perspiration, pH changes, or any combination thereof.

56. The device according to claim 50, wherein the selective deformation of the elastic, viscoelastic or plastic member is user-induced.

57. The device according to claim 56, wherein the user-induced selective deformation is manual, automatic, responsive or semi-automatic.

58. The device according to claim 50, being for use in combination with a prophylactic dressings, bandages, patches, garments, underwear or gels or for use in combination with administration of at least one pyruvate.

59. The device according to claim 58, wherein the at least one pyruvate is contained within any skin-contacting element or region of the device.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0172] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0173] FIGS. 1A-C depict various stretching devices according to the invention. FIGS. 1A and 1B depict a three-dimensionally printed cell stretching device (complete view FIG. 1B) for application of variable radial stretching levels to in vitro cells in culture. As shown in FIG. 1A, the device consists of a bottom plate on which 6 vertical cylinders are placed. A stretchable-substrata plate with 6 sample wells is placed on the bottom plate and the cylinders and under a top plate that is moved downwards to induce stretching of the 6-well plate substrata on the cylinders; distance between the top and bottom plates is controlled by screws and top plate is strengthened by flat plates at the screw locations. FIG. 1C depicts a schematic representation of a prototype device for application of low-level stretching to the in vivo skin regions and deeper tissues through the skin. The exemplary device includes (1) a ring-like, skin-adhesive, stretchable (e.g. elastic) material; (2) stretching element, applying stretching by mechanical, magnetic, magneto-mechanical or electrical means. This element may be a single elastic strip placed between the rings as indicated, or any other configuration that is continuous or non-continuous, circular/annular or any other shape; (3) a ring-like, skin-adhesive, non-stretchable (e.g. stiff and elastic) material that will serve as physical anchor to define the maximal applicable stretching to prevent application of stretching to damage-potential levels.

[0174] FIG. 2 shows an experimental protocol for evaluating cell membrane permeability following damaging tensile-strain. Cells were cultured on elastic substrates for 4 days in growth media (with 0 or 1 mM Sodium pyruvate). In the experiment (starting t=0), cells were pretreated with different sodium pyruvate concentrations (0, 1 and 5 mM) for 4 hours and then sustained 12% strain (or 0% control) was applied for 3 hours. In the last 30 min of stretching 10.sup.−4 M FITC-labeled 4 kDa dextran were added to half of the wells (unstained cells provide autoflourescence blank). Cells were then collected and dextran uptake was measured using flow cytometry relative to unstretched control. Viability was determined by calcein staining, by flow cytometry gating and by cell counting.

[0175] FIGS. 3A-C provide fluorescent distribution curves obtained from FACS system for C2C12 myoblasts cultured in 0 mM NaPy (NaPy free) growth media prior to an experiment; similar plots were obtained for the NIH3T3. The results demonstrate the effect on membrane permeability for pre-treatment with (FIG. 3A) 0 mM, (FIG. 3B) 1 mM, and (FIG. 3C) 5 mM NaPy. The two curves on the left of each panel are strained and unstrained controls (without fluorescent dextran, dashed line shows that those are indistinguishable). The two curves on the right of each panel correspond to unstrained (left) and strained (right) conditions, where reducing distance between then as NaPy pre-treatment concentration is increased shows reduction in strain-damage induced membrane poration.

[0176] FIGS. 4A and 4B show an increase in fluorescence intensity due to uptake of FITC-labeled 4 kDa Dextran into (FIG. 4A) C2C12 myoblasts (FIG. 4B) NIH-3T3 fibroblasts, both pre-treated with Sodium pyruvate (0, 1 and 5 mM) for 4 hours prior to exposure to 12% sustained stretch for 3 hrs. Cells were grown in growth media (GM) without Sodium pyruvate (light) or with 1 mM Sodium pyruvate (dark). Error bars are standard errors. Statistical significance was tested, where *p<0.03, ** p<0.0002.

[0177] FIGS. 5A-D depict kinematics of gap closure of C2C12 myoblasts (left, A and C) and NIH3T3 fibroblasts (right, B and D) for varying stretching levels and sodium pyruvate media-supplement. (FIGS. 5A-B) Normalized maximum migration rate; (FIGS. 5C-D) Time for closure of 90% of initial gap area. Asterisks mark statistically significant results (p<0.05) as compared to values with the same stretching level or with the same sodium pyruvate concentration, in the same cell type.

[0178] FIGS. 6A and 6B depict a three-dimensional (3D) finite element model of the buttocks. Axial cross-section (FIG. 6A) of the mesh with an enlarged tilted view (FIG. 6B) shows the elements distribution at the skin and fat tissue layers closest to the mattress.

[0179] FIGS. 7A-7C depict the damage score threshold 3D plots for (FIG. 7A) Epidermis, (FIG. 7B) Fat and (FIG. 7C) Muscle tissue, caused by applied pressure and temperature for varying durations, reconstructed using the equations developed by Kokate et al. (Wounds 9(4): 111-121 (1997). The plotted surface represents a unity damage score, a value which was determined by the original authors as the damage threshold.

[0180] FIGS. 8A-D demonstrate the development of a computational modeling framework used to conduct studies simulating release of sodium pyruvate (NaPy) from a novel prophylactic dressing: (FIG. 8A) Optical coherence tomography (OCT) image of the sacral skin of a 67 years-old female which has been used for the OCT-based modeling. (FIG. 8B) The four anatomical model variants, representing four levels of skin roughness (R1-R4). (FIG. 8C) Zoom-in on the stratum corneum (SC) and epidermis layers of the skin. The roughness criterion was set as the vertical distance between the top of the highest fold to the bottom of the lowest valley. (FIG. 8D) The thin-slice transient finite element computational model of NaPy diffusing from the dressing (set as an infinite reservoir here) into the skin.

[0181] FIG. 9 is an example of a time course of a change of concentration of sodium pyruvate (NaPy) molecules entering into the depth of the skin (in model variant R2).

[0182] FIGS. 10A-D provide average sodium pyruvate (NaPy) concentrations recorded during 16 hours of simulated use of the novel NaPy-releasing dressing, in each of the model variants representing four levels of skin roughness (R1-R4), in (FIG. 10A) the stratum corneum and epidermis layers, 1 mm (FIG. 10B), 2 mm (FIG. 10C), and 3 mm (FIG. 10D) deep into the dermis.

[0183] FIG. 11 shows time from dressing application to sodium pyruvate (NaPy) concentrations reaching a steady-state, for the four skin roughness levels (R1-R4), in the stratum corneum and epidermis, and 1, 2, or 3 mm deep into the dermis. Steady-state has been defined to occur when NaPy concentration was within ±10% of the plateau NaPy concentration value.

DETAILED EMBODIMENTS OF THE INVENTION

[0184] To evaluate the efficacy of sodium pyruvate (NaPy) as a cytoprotectant material for pressure ulcer prevention, two cell functions were evaluated: (a) changes in the plasma membrane permeability of cells following damage caused by static, damage-level (12%) stretching; those indicate initial damage to cells leading to death, and (b) effects of sodium pyruvate in combination with low-level stretching (3-6%) on the rate of closure of microscale gaps; those were evaluated in the context of prevention. Closure of microscale gaps can prevent the enlargement of gaps into wounds. Both damage-level and low-level stretching were applied using a custom-made radial cell stretching device. Cell experiments were performed on NIH-3T3 fibroblasts (connective tissue and skin cells) and C2C12 myoblasts (muscle precursor cells). The C2C12 are undifferentiated muscle cells, which are the migratory fraction of cells that can migrate to close any micro-damage. A cell-damaging load of 12% stretching was selected based on Slomka N and Gefen A (Ann. Biomed. Eng. 40(3), 606, 2012) and was applied using a custom radial cell-stretching device (Toume, et al., J. Biomech. 49: 1336 (2016)).

Cell Culture Protocols

[0185] Cell culture: undifferentiated C2C12 myoblasts (CRL1722, ATCC, VA) or NIH3T3 cells (CRL-1658) were cultured (separately) in a growth media containing Dulbecco's Modified Eagle's medium (with 4.5 g/L glucose,) 4mM L-glutamine, 1% and Penicillin G+Streptomycin, and 10% or 20% fetal bovine serum (FBS) was respectively added to the C2C12 and the NIH3T3 (all from Biological Industries, Beit-Haemek, Israel). In addition, effects of sodium pyruvate were evaluated in growth media supplement on both cell types: adding 1 mM NaPy as typical media supplement to enhance cell proliferation or growing cells in NaPy free media as expected in physiological conditions in the body; NaPy levels in human blood serum are 0.090-0.12 mM (O'Donnell-Tormey, et al., J. Exp. Med. 165: 500-514 (1987)). Thus, damage-testing was performed on cells that had been cultured in growth media that is supplemented with 0/1 mM NaPy for several days prior to the experiment, respectively, for physiological relevance and for typical cell culture conditions. Cells were incubated at 37° C., and 5% CO.sub.2 and were passaged when confluent (˜80%) every 3-4 days. The cells used for all experiments were always at passages under 30.

Cell Stretching Device

[0186] Radial stretching was applied to an elastic substratum on which cells are cultured. Cells were cultured on a bioflexcell collagen-coated 6-well plate (Flexcell Inc., North Carolina) that was placed between two rigid detachable frames (20×11×6 cm.sup.3) and on top of 6 centrally located vertical cylinders (FIG. 1). The top frame, which was also a cover to the sterile bioflexcell plate, was pushed downwards towards the bottom frame, inducing contact between the elastic substrata and the vertical cylinders. When pushed down the elastic substrata was stretched radially, and the strain was determined by the distance between the top and bottom plates. To maintain a static (constant) strain in the substrata a set of rigid spacers were used to define the distance and screws to reach and maintain it.

Sodium Pyruvate Pre-Treatment Reduces Cell-Membrane Permeability and Cell Death Resulting from Damage-Level Strains

Membrane Permeability Experimental Protocol

[0187] For each experiment, 300,000 cells were cultured 24 hours in parallel on 2-4 bioflexcell collagen-coated 6-well plates (Flexcell Inc., North Carolina). On the day of the experiment the cell growth media (with 0/1 mM NaPy) was replaced with media supplemented with different NaPy concentrations (0, 1 or 5 mM) and cells were incubated for 4 hours; this constitutes the pre-treatment of the cells. A four-hour pre-treatment was chosen as that is a clinically relevant time-scale during preparation e.g. before planned surgical procedures. A flow diagram of the experimental protocol is provided in FIG. 2.

[0188] Each 6-well plate contained two wells with each of the three NaPy pre-treatment concentrations. After 4 hours, all but one of the bioflexcell plates were simultaneously subjected to damaging-level, sustained strain-loads of 12% radial stretching for 3 hours and one plate in each experiment was not stretched for control. After 2.5 hours of stretching, 0.1 mM of fluorescein isothiocyanate (FITC)-labeled 4 kDa Dextran (Sigma-Aldrich, Israel) was added to one of the wells of each NaPy pre-treatment concentrations for the remaining stretch-loading duration of 30 min; the low molecular weight Dextran molecule can pass through small membrane-pores providing a sensitive measure for initial poration. Following the incubation period, the substrate stretching was relieved and cells in all wells were kept in their media for an additional 10. Cells were then rinsed twice with PBS to remove any non-internalized fluorescent stain and cell debris. Then adhered cells were detached with trypsin and suspended in serum-containing growth media to inhibit trypsin activity. Next, cells were centrifuged (900 rpm for 5 min) and resuspended in 0.5 ml of PBS within 12×75 mm polystyrene test tubes for use with fluorescence activated cell sorting (FACS).

[0189] Permeability of the plasma membrane was quantified by cell uptake of the small (4 kDa) FITC-labeled Dextran. Fluorescence intensity changes due to internalized FITC-labeled Dextran and auto fluorescence of over 20,000 cells per sample was measured using a fluorescence-activated cell sorting (FACS) Calibur system (BD Biosciences, New Jersey) with the fluorescence channel. The forward scatter vs. side scatter density plots were also obtained and careful gating was applied to segment populations of live cells for further analysis, i.e. by excluding cell debris and cell-doublets. For each pre-treatment (and growth media) condition 4 measurements were collected: strain-loaded cells with or without added dextran, and unstrained control cells with or without dextran; samples without dextran provide a control for native cell auto-fluorescence (AF). The percent change in dextran uptake is calculated by the mean value of the strained sample relative to the unstrained samples, after subtracting the auto-fluorescence for each.

Membrane Permeability Experiment Results

[0190] Using murine fibroblast (NIH3T3) and myoblast (C2C12) cell lines as a models, respectively, for superficial and deep tissue damage, the effect of NaPy on the cells' ability to maintain the integrity of its plasma membrane under damaging strains was demonstrated. The cells were pretreated with varying levels of NaPy for 4 hours and then applied sustained, radial stretching at damage-level of 12% strain for 3 hours (FIG. 2). Membrane permeability was measured by the uptake of a small (4 kDa) fluorescent Dextran, comparing pre-treated cells with untreated cells and unstrained negative controls; uptake was quantified via FACS.

[0191] FIG. 3 shows representative results of effects of 12% stretching (strain-loading) and NaPy pre-treatment concentration (0/1/5 mM) on the plasma membrane permeability. It was noted that the auto-fluorescence of strained and unstrained controls (without dextran marker) are indistinguishable; this is demonstrated in the C2C12, yet occurs consistently in both cell types and under both growth media NaPy concentrations (0/1 mM). Auto-fluorescence is an indirect indication of morpho-functionality of cells, hence, at this early stage of cell damage the membranes may become porated and homeostasis disrupted, yet the cell structure is still intact. Uptake of FITC-labeled small-molecule dextran was used to identify changes in membrane permeability; the diameter of the 4 kDa dextran marker has previously been shown to be 2.6 nm. Unstrained cells exhibit a measurable natural uptake of the small molecule that is similar under all NaPy pre-treatment concentrations. The natural uptake in unstrained cells serves as the baseline signal, related to natural homeostatic conditions. An increased dextran internalization (higher signal) into cells that are strained to damaging-levels (12%) was observed, especially without NaPy pre-treatment. When cells are pre-treated with increasing concentrations of NaPy, proportionally reduced uptake (a left-shift of the curves) was observed into the strained cells bringing the measured uptake closer to the respective baseline. Specifically, when no NaPy pretreatment was added prior to straining (0 mM NaPy in FIG. 2) an increased uptake of dextran (FIG. 3A) was observed, as compared to NaPy pre-treated cells (FIGS. 3B and C). To evaluate effects of the experimental parameters (i.e. cell type, NaPy in growth media, NaPy pretreatment concentration) on changes in membrane permeability following strain-loading the fluorescent dextran uptake was normalized (see methods) relative to the baseline while subtracting the auto-fluorescence (blank) for strained and unstrained samples.

[0192] Pre-treatment of fibroblasts or myoblasts with sodium pyruvate reduces, in a concentration-dependent manner, the strain-damage induced membrane poration and permeability, which are precursors to loss of cell homeostasis and death (FIG. 4). In the C2C12 myoblasts, a reduction was observed in the cell damage-induced uptake of dextran relative to control that is proportional to the NaPy concentration (FIG. 4A); differences in dextran uptake with increased NaPy concentration were statistically significant (p<0.05). That is, with increasing NaPy concentration a reduction in the differences between the 12% strained cells and their unstrained control was noted. It should be noted that when cells have been pre-exposed to NaPy in their long-term growth media, the NaPy pre-treatment protective effect was more significant. For example, when C2C12 cells were cultured in media containing 1 mM NaPy and then pre-treated with 5 mM NaPy prior to damage application, a reduction was observed in the strain-damage induced membrane poration to insignificant levels. That is, in this condition, differences between dextran uptake of the strained cells and the unstrained control cells were indistinguishable. Thus, extended pre-exposure and pre-treatment with NaPy reduces strain-damage induced disruption of plasma membrane integrity. In the NIH3T3 fibroblasts, a similar effect of reduced damage to membrane integrity (reduced uptake relative to control) was observed with increasing concentrations of NaPy pre-treatment.

Low- and Medium-Level Stretching Combined with Sodium Pyruvate Accelerate Migration of Fibroblasts and Myoblasts

Gap Closure Experiments

[0193] To demonstrate the combined effects of NaPy supplement concentration in the media (0, 1, or 5 mM) and low and medium levels of stretching (3-6%), combinations of both parameters were applied from the time of wounding on the rate of gap closure the time-dependent closure of small gaps was monitored. Mouse myoblast or fibroblast cells (1×10.sup.6 per well, previously grown media containing 1 mM NaPy) were seeded 1-3 days prior to performing a stretching experiment in a six-well (31-mm diameter) culture plate with an elastic, 0.51-mm thick, transparent and collagen-coated substrata bottom (Flexcell Inc., Burlington, N.C.). Cells were grown on the stretchable-bottom plate until a confluent monolayer had formed. The plates with cells were then mounted onto our stretching apparatus and radial stretching to tensile strains of 3% or 6% or 0% (no-stretch) control was applied Immediately following stretching, compressive deformation damage was induced in each monolayer using a rigid optic fiber (˜350 μm diameter). Cells at the center of each well were crushed, inducing an approximately circular cell-damage area with varying sizes (0.05-0.5 mm.sup.2). Media was replaced to remove any cell debris and the fresh media contained varying concentrations of sodium pyruvate: 0, 1 or 5 mM. Then the time-dependent gap closure was evaluated to reveal kinematic features The stretching apparatus was mounted in the motorized microscope stage to facilitate for time-lapse imaging of progression of the gap closure. Cell viability throughout the prolonged experiments was ensured by maintaining 37° C., 5% CO.sub.2 and high humidity, using an incubator that enclosed the microscope (Life Imaging Services, Basel, Switzerland).

Imaging and Analysis of Gap Closure

[0194] Time-lapse imaging was performed using a fully motorized, inverted fluorescence microscope (Olympus IX81, Tokyo, Japan) with a custom MATLAB 2012b (The MathWorks, Natick, Mass.) graphical user interface (GUI) module to automatically control lens positioning and collect images of the gap every 10 minutes for up to 24 hours. Images were taken using an XR Mega-10AWCL camera (Stanford Photonics Inc., Palo Alto, Calif.), using a 10×/NA 0.3 long working-distance, air immersion objective lens at a final magnification of 646.8 nm/pixel. A custom algorithm in MATLAB 2012b was used to automatically analyze the time-progression images of the gap area closure and quantify cell migration and gap closure progression, as described in Topman et al. (Micron 51:9-12; 2012); Toume et al. Int Wound J 14:698-703; 2017). Briefly, the time-dependent area was fit to a Richard's function, which is an asymmetric sigmoid, by minimizing the mean squared error. Using the fitted curve, the maximum migration rate was calculated, which is the maximal slope of the area vs. time, Richards fit. The normalized maximum migration rate was obtained by dividing the maximum migration rate by the initial gap area. In addition, the time for 90% gap area closure, which is indicative of the end of the en masse cell migration regime was obtained (Topman et al. 2012; Med Eng Phys 34:225-232).

Statistical Testing in Gap Closure Experiments

[0195] Results of the different conditions were compared using a two-way analysis of variance (ANOVA) for unequal length samples; Statistical analysis was performed in MATLAB. The interaction parameter was found significant, thus the interactions between the sodium pyruvate concentration and the stretching level and were important; in cases where only the interaction parameter the interaction is considered ‘simple’ by typical definitions in statistical analysis. A one-way ANOVA was also performed in conjunction with post-hoc Tukey-Kramer tests to identify statistically significant differences levels of sodium pyruvate for each level of stretching and vice versa. A P-value lower than 0.05 was considered significant.

Results and Discussion of Gap Closure Experiments

[0196] Both tested cell types, C2C12 myoblasts and NIH3T3 fibroblasts were cultured in media with 1 mM sodium pyruvate up to the time of infliction of compressive deformation damage (micro-gap formation) and concurrent application of radial stretching. At that time, media was replaced with media containing 0, 1 or 5 mM NaPy, thus conditions are of concurrent treatment with NaPy (either at 1 or 5 mM) and of stretching following removal of NaPy (1 mM NaPy growth media replaced with 0 mM media during gap closure).

[0197] When cell monolayers are not stretched prior to wounding (i.e. 0% stretching), the gap closure rate is unaffected by the post-injury NaPy concentration (0, 1, or 5 mM) for both the myoblasts and the fibroblasts (FIG. 5). It should be noted that the migration rates of the NIH3T3 fibroblasts are generally slower, yet differences are not statistically significant.

[0198] In both myoblasts and fibroblasts, the beneficial effects of low- and mid-level stretching are highly dependent on the post-injury NaPy concentration; the cells were grown in 1 mM NaPy up to the injury. When NaPy is supplemented (1 or 5 mM) post-injury, gap closure is significantly accelerated specifically when combined with low-level stretching (3%) in damaged myoblast monolayers (FIG. 5). The normalized gap-closure rate of the myoblasts increases from 16.3% area/hr to 20.5 and 23.7 (being a 26% and 46% increase), respectively, when 1 or 5 mM NaPy are supplemented together with 3% stretching; the time to reach 90% gap coverage decreases from 9.2 hrs, respectively, to 7.3 and 6.3 hrs (being a 21% and 31% decrease). In parallel, it was noted that if NaPy is lacking post-injury the applied stretching can compensate and accelerate gap closure in both cell types. That is, in cells that had previously been exposed to NaPy, yet it is not supplemented in the post-injury medium, stretching accelerates gap closure. Specifically, the maximal migration rates of the myoblasts and the fibroblasts (FIG. 5A-B), respectively, increase from 17.7 to 27.4% area/hr and from 14.6 to 21.1% area/hr (being a 55% and 45% increase) under 6% stretching when NaPy is not supplemented post-injury. In the myoblasts, the acceleration of cells with no NaPy supplement is only statistically significant with 6% stretching. In contrast, in the fibroblasts the acceleration is proportional to the stretching level, at 3% and 6% stretching, respectively, increasing from 14.6% area/hr under 0% stretching to 17.7 and 21.1% area/hr (being a 21% and 45% increase); differences are statistically significant with p<0.05.

[0199] Specific combinations of low and medium-level stretching with exogenous NaPy supplement induced a marked increase in gap closure rate. For both the fibroblasts and myoblasts, when no NaPy supplement was provided post-injury to cells that had previously been exposed to it, the mid-level stretches (6% strain) compensated for deficiency in exogenous NaPy after injury and gap closure was accelerated in a statistically significant manner Importantly, in the myoblasts the smallest evaluated strain (3%) combined with post-injury exogenous NaPy supplement successfully accelerated gap closure in a concentration dependent and statistically significant manner As noted, in the fibroblasts, the pre-damage exposure to low levels of NaPy (1 mM) was sufficient, when combined with low- or medium-level stretching (respectively, 3% or 6%) to accelerate gap closure in a statistically significant manner Lower stretching levels are preferable in the long term, as they reduce the risk for mechanical damage. Hence, the “sweet spot” combination of low stretching levels (e.g. the 3% used here) with low levels of exogenous NaPy (1 mM) supplement provides an optimized treatment protocol for gap closure acceleration.

[0200] In the experiments performed in the gap closure study, the focus is on microscale gaps that may be used as a simplified model for damage repair or, for example, represent the actual scale of damage caused during initiation of pressure ulcers (pressure injuries), given that the initial stage of pressure ulcer formation includes death of small groups of cells. In this context, the sites of mechanical deformation-induced damage can be in some cases be foreseen and depend on patient anatomy and length of immobility period, such as when a person is anesthetized at a certain body posture in preparation for surgery. In our experiment, the stretching and the media replacement are applied together with the injury, effectively performing the “wounding” and the “treatment” (cleaning of cell debris and related signaling molecules, NaPy supplement, applied stretching) at the same time. For the example of pressure ulcer initiation, in performing the stretching immediately prior to cell death, a condition of pre-treatment is in fact simulated prior to or immediately following initial cell damage, in a preventative approach; closure of small gaps will prevent the cascade of further damage development.

Measuring Subepidermal Moisture to Detect Pressure Injury

[0201] Cell death at the onset of a PU triggers inflammatory processes that as a side-effect lead to blood plasma fluids escape from the vasculature, resulting in microscopic onset of edema. The volume of the fluids builds up gradually in the tissue, eventually forming edema. As more immune cells are recruited to the cell death site, the process that begins microscopically progresses to a larger scale. A commercial technology to measure the change in fluid content, i.e. the subepidermal moisture (SEM) has recently been introduced in the form of a SEM Scanner (Bruin Biometrics Europe) which provides a clinically indicative biophysical biomarker for early stages of PU formation. The SEM scanner is a hand-held device sensitive to changes in the biocapacitance of the affected soft tissues, or their resistance to transmission of non-damaging electrical fields. Specifically, gradual accumulation of fluids in tissues make the tissues progressively less resistant to electrical fields, and hence the relative permittivity (dielectric constant) of the tissues increase from that of the health tissues towards that of water. The SEM Scanner is sensitive to small changes in the amounts of extracellular fluids, and provides standardized, objective and quantitative measures for detecting changes associated with the edema build-up as a reaction to the immune response to the death of the first cells. There is a substantial volume of published literature demonstrating that the SEM Scanner can detect subdermal tissue damage 3-10 days before it is visible to the naked eye (See for example: (a) Bates-Jensen B M, McCreath H E, Nakagami G, Patlan A. Subepidermal moisture detection of heel pressure injury: The pressure ulcer detection study outcomes. Int Wound J. 2018 April; 15(2):297-309; (b) Bates-Jensen B M, McCreath H E, Patlan A. Subepidermal moisture detection of pressure induced tissue damage on the trunk: The pressure ulcer detection study outcomes. Wound Repair Regen. 2017 May; 25(3):502-511; (c) O'Brien G, Moore Z, Patton D, O'Connor T. The relationship between nurses assessment of early pressure ulcer damage and sub epidermal moisture measurement: A prospective explorative study. J Tissue Viability. 2018 in press, available online, doi: 10.1016/j.jtv.2018.06.004; (d) Gefen A, Gershon S. An Observational, Prospective Cohort Pilot Study to Compare the Use of Subepidermal Moisture Measurements Versus Ultrasound and Visual Skin Assessments for Early Detection of Pressure Injury. Ostomy Wound Manage. 2018 September; 64(9):12-27).

Computer Simulations in Human Anatomy:

[0202] A three-dimensional finite element (FE) model of the human anatomy was developed (FIG. 6), which considers the ‘microenvironment’ (temperature and humidity) and applied simulated-deformation (initially compression) due to mattresses. An MRI based buttocks FE model was used and several predictive tools were incorporated to simulate how microclimate affects tissue biomechanically and biothermally in macroscale when in the patient is in bed.

[0203] The results of this model are temperature, strain and damage score maps of the 3D buttocks model under different conditions, which are shown in FIG. 5. A higher damage threshold on the epidermal layer as compared to fat and muscle was observed. For all three tissue types, the threshold for damage was temperature dependent, exponentially increasing over 40° C. (FIG. 7).

Delivery of Sodium Pyruvate (NaPy) from an Active Dressing to Protect Sacral Skin and Underlying Tissues

[0204] Four finite element computational model variants of the sacral skin were developed, representing four levels of skin roughness. These model variants were used to investigate how prophylactic dressings, pre-loaded with NaPy, can be used for transdermal delivery of NaPy, for improving skin and subcutaneous tissue tolerance to sustained deformation exposures and hence, tissue resistance to PU development. For this purpose, application of such novel dressings loaded with a known NaPy concentration was modeled and monitored simulated free NaPy diffusion into the skin for a period of 16 hours.

[0205] Optical coherence tomography (OCT) images of the sacral skin were used to generate four two-dimensional anatomical skin models, representing different levels of skin roughness (FIGS. 8A-B). Two-dimensional images were acquired with a scan length of approximately 5 mm, a lateral resolution of up to 8 μm, and a maximum penetration depth of ˜1.5 mm Segmentation software (the ScanIP module of Simpleware®) was used to segment the different layers of the skin. A roughness criterion (R) was set as the maximal vertical distance between the top of the highest fold and the bottom of the lowest valley of the stratum corneum (SC), and the four areas of interest were chosen so that R.sub.1=20 μm, R.sub.2=30 μm, R.sub.3=40 μm, and R.sub.4=50 μm (FIGS. 8B-C; R.sub.1 and R.sub.2 are from the same subject, and likewise, R.sub.3 and R.sub.4 are from the same other individual). Then, a constant minimal thickness was assigned to the anatomical model variants so that each of the variants was 1×3×0.05 mm and included the SC, epidermis and dermis layers (FIG. 8B). Next, a geometrical representation of a flat dressing was added to each of the model variants, and positioned as close as possible to the rough SC surface (FIG. 8D).

[0206] The dressing and skin layers were considered as biphasic-solute materials in order to be able to simulate the coupled structural response of skin to the (bodyweight) loading and the diffusion of the NaPy released from the dressing as it compresses onto the skin (under the bodyweight forces). Physical and diffusional properties of the skin layers, dressing and NaPy molecules were adopted from the literature. Specifically, NaPy molecules were assigned a molecular weight of 110 g/mol and a neutral chemical charge. Solubility, diffusivity and free diffusivity of NaPy molecules in the aqueous phase of the dressing and tissues were considered isotropic and set at 100 mg/mL, 0.0005 mm.sup.2/s and 0.001 mm.sup.2/s, respectively.

[0207] The dressing and skin layers were assigned a solid volume fraction of 0.2 and constant isotropic permeability characteristics as follows. Dermal permeability coefficient (K.sub.p) of NaPy was calculated using:

log K.sub.p=−2.72+0.71.Math.(log Ko/W)−0.0061.Math.(MW)

[0208] where Ko/W=−5.05 and MW=110.04 g/mol.

[0209] Since the skin barrier properties are mostly attributed to the SC layer, and given that transepidermal water loss (TEWL) increases 20-fold when the SC is removed, the permeability coefficients of the epidermis and dermis were set as 20-times greater than that of the SC. The final K.sub.p values of skin were set at 2.9322.Math.10.sup.−10 mm/s for the SC and 5.86445.Math.10.sup.−9 mm/s for the epidermis and dermis. The K.sub.p of the dressing was set at 0.001 mm/s.

[0210] Constitutive laws and mechanical properties of the model components were also adopted from the literature. Specifically, the dressing material was assumed to be isotropic linear-elastic material with an elastic modulus of 19 kPa and a Poisson's ratio of 0.3. The skin layers were assumed to be nearly incompressible (Poisson's ratio of 0.49), non-linear isotropic materials with their large deformation behavior described using an uncoupled Neo-Hookean material model with a strain energy density (SED) function W:

[00001]$\begin{array}{cc}W=\frac{G_{\mathrm{ins}}}{2}\left({\lambda }_1^2+{\lambda }_2^2+{\lambda }_3^2-3\right)+\frac{1}{2}{K\left(\ln J\right)}^2& \left(1\right)\end{array}$

where G.sub.ins is the instantaneous shear modulus, λ.sub.i (i=1, 2, 3) are the principal stretch ratios, K is the bulk modulus and J=det(F) where F is the deformation gradient tensor. The instantaneous shear moduli assigned to the skin layers were 839 kPa, 352 kPa and 7.55 kPa for the SC, epidermis and dermis, respectively.

[0211] Boundary conditions were chosen to simulate the application of the novel prophylactic sacral dressing loaded with NaPy in a thin-slice model configuration. The dressing was assigned an initial homogenous concentration of 100 mM and the top surface of the dressing was held at a constant NaPy concentration throughout the simulation, which represented the intra-dressing NaPy reservoir. The front, back, bottom and side planes of the dressing and skin layers were assigned zero flux across them, and biphasic-solute contact was defined only between the bottom of the dressing and top of the SC, so that NaPy molecules may only leave the dressing into the skin, where contact is established (but not return to the dressing). The front, back and left planes of the dressing and skin were fixed for perpendicular displacements, while the right surfaces were released to allow adequate convergence of the numerical simulation and relaxation of the osmotic stresses during the diffusional response. The dressing was then lowered by 0.2 mm over two seconds, until contact was established between the dressing and the SC, and NaPy molecules were allowed to translate by diffusion from the dressing into the skin, over a period of 16 hours of simulated application (FIG. 9).

[0212] Meshing the model variants was again performed by means of the ScanlP® module of Simpleware®, using 4-node linear tetrahedral elements (FIGS. 8B-C). Each model included approximately 1,700 elements describing the dressing, 1,550 elements describing the SC, 1,840 elements describing the epidermis and 8,250 elements describing the dermis.

[0213] Simulations were set up in PreView of FEBio (Ver. 1.19), analyzed using the Pardiso linear solver of FEBio (http://mrl.sci.utah.edu/software/febio) (Ver. 2.5.0) in its biphasic-solute transient mode, and post-processed using PostView (Ver. 1.10). A 64-bit Windows 8-based workstation with 2×Intel Xeon E5-2620 2.00 GHz CPU and 64 GB of RAM was used for solving the coupled structural-diffusion problems of the NaPy release from a prophylactic dressing.

[0214] The transient average effective NaPy concentrations in the SC and epidermis layers together, and at depths of 1 mm, 2 mm and 3 mm into the dermis were measured for the four examined levels of OCT-measured skin roughness. The times until steady-state has been reached were additionally calculated for each model variant and each examined depth of skin. The time required for convergence of a run was defined as the time from the simulated application of the dressing to the first time point where the NaPy concentration level at the examined depth did not change by more than ±10% with respect to the plateau value.

[0215] An example time course of the NaPy concentration as the substance enters the skin is shown in FIG. 9. During the vertical displacement of the dressing, contact area between the dressing and the SC is established and NaPy molecules begin to diffuse from the dressing into the skin. Diffusion persists until steady-state is reached and NaPy concentration stabilizes, as expected. The average dermal NaPy flux stabilized after ˜5 minutes at 0.35 nmol/cm.sup.2.Math.h.

[0216] NaPy concentrations in the SC and epidermis layers increased rapidly in all the model variants, and has peaked at 6.92 mM, 4.85 mM, 5.3 mM and 3.74 mM in model variants R.sub.1, R.sub.2, R.sub.3 and R.sub.4, respectively, 7-9 seconds post application of the dressing (FIG. 10A). The NaPy concentrations stabilized after 1.15-4.5 hours at 2.98 mM, 2.45 mM, 2.23 mM and 1.34 mM in model variants R.sub.1, R.sub.2, R.sub.3 and R.sub.4, respectively (FIGS. 10A and 11), that is a coefficient of variation (COV) of 30.4% which reflects the micro-anatomical variability and its impact on the variability of the individualized diffusion responses. Across all the examined dermis depths, NaPy concentrations stabilized after 3-6.6 hours (for all micro-anatomies), at values of 2.88 mM, 2.45 mM, 2.16 mM and 1.24 mM in model variants R.sub.1, R.sub.2, R.sub.3 and R.sub.4, respectively (FIGS. 10A and 11), which yields a COV of 31.8% (again pointing to the extent of variability in individual diffusion responses). Correspondingly, it was found that the relative contact areas between the dressing and the SC reached 54.1%, 51.1%, 44.6% and 28.7%, in model variants R.sub.1, R.sub.2, R.sub.3 and R.sub.4, respectively. The latter demonstrates the potential variability in dressing-skin contact conditions, which is a derivative of the individual skin roughness, being yet another factor that influences the individual diffusion pattern of NaPy into the skin.

[0217] Overall, in all but one model variant, NaPy concentrations stabilized faster in the more superficial layers of the skin and slower deeper within the dermis, which could be foreseen given that release is from the NaPy reservoir within the dressing (FIG. 11). For example, in model variant R.sub.2, NaPy concentration reached 90% of its final value after 2.28, 3.96, 5.2 and 5.47 hours, in the SC/epidermis, and 1, 2, and 3 mm deep into the dermis, respectively. Additionally, in all but one examined depths, NaPy concentrations stabilized faster in the model variants that represented smoother skin surfaces. For example, at 2 mm into the dermis, steady-state has been reached after 4.13, 5.2, 4.98 and 6.3 hours in model variants R.sub.1, R.sub.2, R.sub.3 and R.sub.4, respectively where R.sub.1, R.sub.2, R.sub.3 are the smoother skins. As mentioned already, this behavior originates from the quality of attachment of the dressing onto the skin, which typically has less contact area for a more rough skin surface (such as in an aged, wrinkled skin).

[0218] In summary, a multiple OCT-based FE computational model variants was developed and used in order to evaluate the transdermal delivery capacities of novel NaPy-loaded prophylactic sacral dressings in different individuals (patent pending by co-inventor AG). Further, effects of skin roughness were studied on the resulting diffusional response of the NaPy released from the dressing. A steady state NaPy concentrations was found in the dermis, being 1.25-3% of the concentration loaded in the applied dressing, which is in good agreement with published data regarding the absorbance capacity of dermal tissues. Steady state concentrations also found within the dermis were correlated with the relative contact area between the dressing and the SC, which is a direct outcome of the skin roughness level. This effect was to be expected since the greater the contact area available for diffusion of NaPy, the greater the NaPy flux is across the contact area, and the greater its resulted concentrations in deeper tissue layers. Nevertheless, it should be noted that in real-world conditions, the skin roughness under the dressing will likely decrease as the skin temperature and (the associated) level of SC hydration are expected to increase. Altogether, a smoother, warmer and more moist skin under the dressing (compared to bare skin) would result in elevated dermal permeation and hence, increased intra-dermal NaPy diffusion with respect to our present modeling predictions which did not consider these complex skin-dressing interactions at this stage.

[0219] In general, the diffusion of molecules through the skin is mostly attributed to the barrier properties of the SC, and also, strongly, on the size of the diffusing molecule, its hydrophilic or hydrophobic nature, and the applied concentration gradient. The permeability of intact human skin is significantly decreased for diffusants with a molecular weight (MW) above 500 Daltons. The SC, which is only a few micrometers thick, consists of apoptotic keratinocytes surrounded by keratin-rich lipid bilayers. This means that small hydrophilic molecules penetrate through the SC via an intracellular route, or through the hair follicle/sweat glands openings in the SC. Hence, the level of hydration of the SC has a substantial effect on the diffusivity of small hydrophilic molecules, such as NaPy, with greater diffusivity as the hydration level increases. Furthermore, as blood flow in the dermis increases, transport of small hydrophilic molecules increases as well and diffusion to deeper tissues wanes down. The hydration level of the SC as well as the blood flow in the dermis should therefore be considered in future modeling of transdermal delivery from prophylactic dressings.

[0220] In the medical literature, there are only sparse data regarding the permeability of the SC to NaPy. However, pyruvic acid is commonly used topically on the skin (e.g. in the treatment of acne), with obvious potency in deeper skin layers, and hence could be assumed that NaPy is able to penetrate the SC spontaneously and in a similar way. Furthermore, the results of the simulations agree with those obtained by others who examined ex-vivo transdermal delivery of various topical analgesic medications, using Franz Diffusion Cells. Early studies found that the transdermal flux of Diclofenac Sodium which is a similar sodium salt with a MW of 318 g/mol that has been applied on the skin using a 3%-cream reached 0.321-0.943 nmol/cm.sup.2.Math.h after 24 hours. It was also shown that a total of 0.846-1.96% of the applied diclofenac sodium was absorbed in the skin after 48 hours.

[0221] NaPy is a relatively small molecule (e.g. compared to pyruvate acid) and hence it diffuses freely in the aqueous phase of soft tissues. For example, the corneal penetration of NaPy has been studied in living human eyes 2 hours prior to extraction of the corneal tissue due to cataract surgery. The level of NaPy in control tissue samples of patients who did not receive NaPy eye drops was only 0.145+/−0.06 mM (which reflects the natural, baseline corneal NaPy levels, whereas in the group given the NaPy eye drops, it increased to approximately 0.35-0.525 mM. These findings are in good agreement with the results of the present simulations with regard to timeframes for plateau of diffusion and concentration levels at the target tissues, however, the use of prophylactic dressings, unlike the application of eye drops or creams, allows for continuous administration of NaPy molecules. Accordingly, release of NaPy from a prophylactic dressing is able to induce a relatively constant concentration in the target soft tissues over time, which is a considerable advantage when the goal is PUP for an estimated specific time frame, such as during (a certain, known type of) surgery. Additionally, the few hours needed to achieve a steady state potent NaPy concentration in our modeling, make a reasonable timeframe for applying such NaPy-loaded sacral prophylactic dressings prior to a planned, scheduled surgery. For example, if the surgery is to be performed in a supine patient, a NaPy-releasing prophylactic sacral dressing can be applied approximately 4 hours prior to anesthesia (based on the data shown in FIG. 10) to enhance sacral soft tissues tolerance to the sustained deformations caused by bodyweight forces.

[0222] In conclusion, NaPy-releasing prophylactic dressings is capable of improving soft tissue tolerance to sustained deformations. The time needed to achieve steady-state NaPy concentrations in the dermis was approximately 4 hours, which makes the protective effect of such dressings applicable in preparing a patient for surgery, or for use in intensive care units. Individual skin roughness might theoretically affect the resulting NaPy concentration in the dermis, considering microclimate and skin barrier alternations that likely occur under the dressing.