Sodium pyruvate pre‐treatment prevents cell death due to localised, damaging mechanical strains in the context of pressure ulcers

Abstract

We demonstrate sodium pyruvate (NaPy) pre‐treatment as a successful approach for pressure ulcer (PU) prevention by averting their aetiological origin—cell‐level damage and death by large, sustained mechanical loads. We evaluated the NaPy pre‐treatment effect on permeability changes in the cell's plasma membrane (PM) following application of in vitro damaging‐level strains. Fibroblasts or myoblasts, respectively, models for superficial or deep‐tissue damage were grown in 0 or 1 mM NaPy, emulating typical physiological or cell culture conditions. Cells were pre‐treated for 4 hours with 0 to 5 mM NaPy prior to 3‐hour sustained, damaging‐level loads (12% strain). PM permeability was quantified by the cell uptake of small (4 kDa), fluorescent dextran compared with unstrained control using fluorescence‐activated cell sorting (FACS). Pre‐treatment with 1 mM, and especially 5 mM, NaPy significantly reduces damage to PM integrity. Long‐term NaPy pre‐exposure can improve protective treatment, affecting fibroblasts and myoblasts differently. Pre‐treating with NaPy, a natural cell metabolite, allows cells under damaging‐level mechanical loads to maintain their PM integrity, that is, to avoid loss of homeostasis and inevitable, eventual cell death, by preventing initial, microscale stages of PU formation. This pre‐treatment may be applied prior to planned periods of immobility, for example, planned surgery or transport, to prolong safe time in a position by preventing initial cell damage that can cascade and lead to PU formation.

1. INTRODUCTION

Pressure ulcers (PUs) are common in many care settings and at all age groups and have adverse health outcomes and high treatment costs. PUs are initiated by strain‐induced local damage, caused by external loading or occuring at weight‐bearing sites in the body (Figure 1), which can lead to cell death1, 2; individuals with sensory impairment, for example, with neurological damage or under sedation during operation, are unable to self‐relieve those damaging‐level mechanical strains. At those damaging‐strain loaded interfaces, large mechanical strains that are transmitted to cells can cause their death by necrosis or apoptosis.1 For example, at bony prominences, damage can initiate at bone‐muscle interfaces and then spread towards the skin3; that is termed a deep tissue injury (DTI). Damage can also initiate at the skin, from contact with clothing, support and other surfaces,4, 5 or medical and other devices, for example, in paediatric care.6, 7 Prevention is the only cost‐effective approach to avert the dangers of PU formation8 in temporarily/permanently immobilised individuals or patients who often have other underlying medical conditions. Currently, efforts in PU prevention have been focused on the redistribution of potentially damaging mechanical strains and stresses by a variety of specialised support surfaces and dressings9, 10, 11 or by nutritional support and the application of various topical agents and dressings designed mainly to maintain a moisturised and healthy skin.3, 12 Those approaches mainly address macroscopic (organ‐scale) or systemic effects and do not currently actively interact with tissues, especially deeper tissues where, for example, DTIs may initiate. Hence, approaches to avert the formation of the initial mechanical damage to cells1 at the earliest stages are crucial.

Figure 1.

Bodyweight and/or external damaging‐level forces can lead to development of cell damage in the form of plasma membrane poration that leads to loss of homeostasis and eventual cell death. Death of several cells produces micro‐scale gaps in tissue, which are the initial stage that can potentially lead to the development of a pressure ulcer

Mechanical loading at the cell level can produce a wide range of cell‐killing damage1 that, when unchecked, may progress through a cascade of steps13 to form a PU. This cascade starts with microscopic injury where the death of several cells (Figure 1) can progress to tissue‐scale effects at the skin or at deep tissues. Cell death induces a natural inflammatory response that leads to an increase in interstitial pressure, causing further loading on yet‐undamaged cells, which results in a vicious, damage‐increasing cycle over time, leading to PU development.13 It has previously been established that macroscopic loads on tissues are translated to cell‐level deformation, inducing localised tensile strains in cells through their plasma membrane (PM).1, 14, 15, 16, 17 Excess loading, for example, due to bodyweight at bony prominences or loads applied externally to the body, can cause cell death, inducing deformations that disrupt the structure and dynamics of the cytoskeleton,1, 18 leading to the compromised integrity of the PM1, 14, 15, 17 (Figure 1). We have recently shown that, during the currently undetectable, very early stages of microscale damage, that is, when few cells die and microscale gaps form, it is possible to locally repair the formed gaps by applying innovative mechanobiology‐based approaches to accelerate natural cell migration and gap closure.19, 20 The damaging‐deformation‐driven poration of the PM affects molecular transport,15, 16, 17, 21, 22, 23, 24, 25 compromises cell homeostasis, and eventually causes cell death13, 26 at timeframes ranging from tens of minutes to several hours; this is substantially more rapid than the progression of ischaemic damage, which for decades has been mistakenly perceived to be the primary cause of PUs.22 Thus, maintaining the structural integrity of the PM may extend the safe time for deformed cells under potentially damaging strains, that is, the safe time for a patient in a position. With this in mind, we have developed a novel mechanobiology and cell dynamics‐based technology to prevent the formation of PUs at different tissues (skin and deeper) and to concurrently repair any microscale damage that may form.20, 27 Thus, we present an innovative approach to PU prevention based on a profound understanding of the aetiology of initiation of PUs1, 3, 13 at the cellular level and on the understanding of cell mechanobiology and dynamics under various normal and damage states,19, 26, 28, 29, 30, 31, 32 especially in the context of wounds.1, 19, 20, 33 To prevent the initial microscale damage ‐ localised cell death ‐ that initiates the vicious cycle leading to PU development, we evaluate a pre‐treatment with sodium pyruvate (NaPy) to promote cell survival under damaging mechanical loads.

We evaluate pre‐treatment with exogenous pyruvates prior to the induction of damaging‐level strains to support cell homeostasis and viability under those strains. Pyruvates are a precursor of a Krebs cycle intermediate (acetyl‐coenzyme A)34 and are thus part of many key metabolic pathways related to cell respiration and energy production; pyruvates are naturally produced in cells by glycolysis. This has led to the standard use of NaPy as a growth media supplement for many cultured cell lines (typically at 1 mM concentration) to increase cell proliferation rates. Pyruvates are commercially used as food supplements and weight loss aids35 and in skin ointments and treatments36 because of their apparent antioxidant and anti‐inflammatory capacity.37, 38, 39 Aptly, NaPy has been shown to concurrently reduce inflammation and increase healing after viral infection in animals.40 Various uses of pyruvates and specifically NaPy have been described in the context of tissue repair and protection following damage, for example, by trauma, ischaemia, or toxicity. For example, NaPy has been shown to minimise deficits following traumatic myocardial infraction in pigs41, 42 and reduce cortical contusion injury and hypoxic‐ischemic damage in rat brains.43, 44, 45 In addition, NaPy has improved rat renal function after glycerol‐induced renal failure46 and has been proposed as an adjunct treatment to protect fibroblast cell lines from chemotherapy‐induced death.47 Physiological NaPy levels in human blood are low, 0.090 to 0.12 mM,48 and abnormal levels of NaPy in blood serum and extracellular spaces are often an indicator of disease.49 NaPy has been suggested to add to the available energy stores of tissues and cells26 during periods of injury‐induced, increased metabolic demands.43 Considered together, these studies highlight a potential application of pre‐treatments with NaPy (prophylactically) applied prior to damage induction, which may prevent cell death under damaging loads,1, 26 thus averting the early stage of PU formation (Figure 1).

Here, we present, for the first time, an innovative approach to dynamically protect cell integrity and maintain its homeostasis at susceptible tissue sites by pre‐treating prior to expected damaging‐level loading that can lead to strain‐induced cell damage and death.1 We have recently shown that low‐level (non‐damaging) strains,19 especially combinations of non‐damaging low‐level strains with NaPy supplement,20 accelerate the closure of micro‐scale gaps caused by death of 100's of cells. We propose pre‐treatment with pyruvates to be applied prior to expected exposure to damaging‐level strains as an approach to facilitate dynamic cell responses26 to expected loading, thus preventing potential cell damage and death and, for example, extending the safe time in a position. We utilise NaPy as an innovative prophylactic pre‐treatment to prevent single cell‐level damage due to mechanical loading. Damaging‐level mechanical loading has been shown to disrupt the integrity of the cells' plasma membrane15, 17 that can affect cell homeostasis and viability under strain.50 We thus evaluate changes in PM poration induced by damaging‐level strains by varying concentrations of NaPy pre‐treatment. Specifically, we compared the uptake of a small (4 kDa), fluorescent dextran into cells that had been pre‐treated for 4 hours with NaPy prior to the application of damaging‐level (12%) strain for 3 hours to untreated control cells; the experiment times represent typical times, for example, for scheduled, non‐emergency surgery. We concurrently compare conditions where the growth media of cells include NaPy or are NaPy free prior to the experiments to demonstrate the effects of extended exposure to NaPy. Our results show that pre‐treatment and extended exposure to sodium pyruvate prior to mechanical damage facilitate the maintenance of the PM integrity of cells under damaging‐level strains.

2. METHODS

2.1. Cell culture

We used undifferentiated C2C12 murine myoblasts (CRL1722, American Type Culture Collection (ATCC), Manassas, Virginia) and NIH3T3 murine embryonic fibroblasts (CRL‐1658), both established cell lines typically used in in vitro PU formation studies,22 of susceptible superficial and deep tissue sites. These cells provide simplified models for the cell types prevalent at susceptible superficial and deep tissue sites, which would be involved in the PU formation process. The undifferentiated cells are also migratory and would subsequently be able to migrate to repair any micro‐scale damage that may form.51

Cells were cultured (separately) in growth media containing Dulbecco's Modified Eagle's medium (DMEM) with 4.5 g/L glucose and without added pyruvate (pyruvate free), with 10% or 20% foetal bovine serum (FBS), respectively, and with 4 mM L‐glutamine and 1% and Penicillin G + Streptomycin (all from Biological Industries, Beit‐Haemek, Israel). To evaluate the effects of sodium pyruvate media supplement in the growth media, two conditions were tested: 1 mM NaPy or 0 mM NaPy‐free. The former (1 mM NaPy) is a typical media supplement in cell culture, intended to enhance cell proliferation, and the latter (NaPy free) is close to physiological conditions in the body as NaPy levels in human blood serum are 0.090 to 0.12 mM.48 Mechanical loading effect experiments were performed on cells that had been cultured in growth media supplemented with 0 or 1 mM NaPy for several days prior to the experiment. Cells were incubated at 37°C and 5% CO₂ and were passaged at about 80% confluence every 3 to 4 days. For all experiments, cells were used only up to 30 passages from ATCC stock.

2.2. Cell stretching device

Damaging‐level deformations were applied through a custom, three‐dimensionally printed device52 (Figure 2) by radially stretching cells that had been grown to confluency on an elastic substratum. Radial stretching or straining of the cells is appropriate in the context of PUs. PUs form primarily because of compressive forces;53 however, those are translated to a compound loading mode in tissues and cells; the loading mode differs across dimensional scales. Compression applied at the mesoscale to tissues will be shared between the extracellular matrix (ECM) and the embedded cells. As ECM is typically stiffer than the cells, tissue compression will induce large stretching deformations in cells as previously shown by computational simulations.14 In the current experiments, mechanical strains are applied directly to the cells (ie, not through an encompassing ECM, which is not part of our model system). Hence, radially applied strains emulate the dominant type of strains that are typically experienced by the cells during tissue compression.

Figure 2.

Design of cell‐stretching device for variable, controlled stretching levels. (A) View of the separated cell‐stretching device elements. Cells are seeded in a 6‐well plate with a stretchable substrate that is lowered onto round inserts, causing radial stretching; the stretching is directly transmitted from the substrate to the adhered cells. The device parts are screwed together. (B) The fully constructed cell‐stretching device contains a stretchable‐substrate 6‐well plate and the stretching level is controlled through the distance between the top and bottom plates, where the screws are used to move the plates closer and increase the strain

Cells were cultured on a bioflexcell collagen‐coated 6‐well plate (Flexcell Inc., Burlington, North Carolina) that is placed between two rigid, detachable frames (20 × 11 × 6 cm³). The top frame serves as the plate cover to maintain sample sterility. The bottom frame holds six vertical, hollow cylinders that are placed centrally in each of the six wells (Figure 2). The distance between the top and the bottom frames is controlled by screws, where moving the frames closer pushes the elastic substrata of the 6‐well bioflexcell plate into contact with the vertical cylinders; a set of rigid spacers facilitates reproducible application of the same strains in all experiments. Contact with the cylinders induces radial stretching in the elastic substrata, with the strain level being determined by the distance between the top and the bottom plates. The percentage strain is determined relative to non‐stretched substrata; these substrata were previously determined to be linearly elastic up to strains of 18%.17

2.3. Stretching experiment protocol

For each experiment, 300 000 cells/well were cultured for 24 hours on two to four bioflexcell 6‐well plates. On the day of the experiment, the cell growth media (with 0 or 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 4‐hour pre‐treatment was chosen as that is a clinically relevant time scale during preparation, for example, before planned surgical procedures.

A flow diagram of the experimental protocol is provided in Figure 3. Each 6‐well plate includes three NaPy pre‐treatment concentrations (0, 1, or 5 mM) with two repeats each. After 4 hours, all but one of the bioflexcell plates were subjected to damaging‐level, sustained strain loads of 12% radial stretching (as determined in17) for 3 hours, and the last plate served as unstrained control. After 2.5 hours of stretching, 0.1 mM of fluorescein isothiocyanate (FITC)‐labelled 4 kDa dextran (Sigma‐Aldrich, Israel) was added to one of the two (repeat) wells of each NaPy pre‐treatment concentration for the remaining stretch‐loading duration of 30 minutes, including the unstrained control plate; the low‐molecular‐weight dextran molecule can pass through small PM pores,15 providing a sensitive measure for initial poration; the 4 kDa dextran marker has previously been shown to be 2.6 nm in size.54 After 3 hours, the substrate stretching was released, and cells in all wells were maintained in their media for an additional 10 minutes to allow extra dye to enter stable pores (increase signal) and to distinguish between rapidly repaired pores that could affect permeability changes.55 Cells were then rinsed twice with phosphate‐buffered saline (PBS) to remove any non‐internalised fluorescent stain and cell debris. The washed cells were detached with trypsin and suspended in serum‐containing growth media to inhibit trypsin activity. Next, cells were centrifuged (900 rpm for 5 minutes) and re‐suspended in 0.5 mL of PBS within 12 × 75 mm polystyrene test tubes for use with fluorescence‐activated cell sorting (FACS).

Figure 3.

Experimental protocol. Three 6‐well plates were seeded with 3 × 10⁵ cells and cultured overnight. The growth media (0 or 1 mM sodium pyruvate [NaPy]) was replaced by media supplemented with different NaPy concentrations (0, 1, or 5 mM) and incubated for 4 hours. Then, two plates were exposed to sustained damaging‐level load (12% radial strain) for 3 hours, and one was used as unstrained control. During the last 30 min of loading, fluorescein isothiocyanate (FITC)‐labelled 4 kDa dextran was added to the media of half the wells (labelled with “D”). Following the experiment, cells were trypsinised, centrifuged, and suspended in 0.5 mL of phosphate‐buffered saline (PBS) for fluorescence‐activated cell sorting (FACS) analysis

2.4. Flow cytometry

Permeability of the plasma membrane was quantified by cell uptake of the small (4 kDa) FITC‐labelled dextran. Fluorescence intensity changes because of internalised FITC‐labelled dextran and auto‐fluorescence (AF) of over 20 000 cells per sample was measured using an FACS Calibur System (BD Biosciences, New Jersey) with the fluorescence channel (excitation/emission: laser 488/530 nm). The forward‐scatter versus side‐scatter density plots were also obtained, and careful gating was applied to segment populations of live cells for further analysis, that is, by excluding cell debris and cell doublets.

For each NaPy concentration in pre‐treatment and growth media, we collect four measurements: strain‐loaded cells with and without added dextran and unstrained control cells with and without dextran; samples without dextran (strained and unstrained) provide a control for native, cell AF, used as the background for subtraction. The fluorescence intensity of each sample/control was calculated as the geometric mean of the fluorescent intensity histogram of the sample (as typically accepted in FACS analysis), using the FCS Express 6 Flow (De Novo Software). The dextran uptake of strained relative to control samples was then calculated with the geometric means, subtracting from each the geometric means of the AF specific for each combination (combo) of NaPy concentration in the growth media and in the pre‐treatment (see application in Figure 5) as follows:

$$\text{Dextran uptake}\left[\%\right]=\frac{{\text{Strained}}_{\mathit{\text{NaPy combo}}}^{\mathit{\text{Dextran}}}-{\text{Strained}}_{\mathit{\text{NaPy combo}}}^{\mathit{AF}}}{{\text{Unstrained}}_{\mathit{\text{NaPy combo}}}^{\mathit{\text{Dextran}}}-\mathrm{Un}{\text{strained}}_{\mathit{\text{NaPy combo}}}^{\mathit{AF}}}\times 100-100.$$

Figure 5.

Plasma membrane permeability (fluorescein isothiocyanate [FITC]‐dextran uptake) under varying amounts of sodium pyruvate (NaPy) in pre‐treatment. (A) C2C12 myoblasts and (B) NIH3T3 fibroblasts were grown in growth media (GM) containing either 1 mM (blue) or 0 mM, NaPy‐free media (grey) and then pre‐treated for 4 hours with different NaPy concentrations (0, 1, or 5 mM). The change in cell uptake of FITC‐labelled 4 kDa dextran because of 12% strain applied for 3 hours relative to unstrained cells was measured using FACS; see methods for detailed explanation of the ratio calculation. Error bars are standard errors around the mean, and asterisks mark statistical significance (* P < 0.03 and ** P < 0.0001); in C2C12, statistically significant differences between all cases of 0 and 1 mM growth media are not marked for clarity

2.5. Statistical testing

We performed four biological repeats on different days with three triplicates each for technical repeat (i.e., N = 12) for each cell type under each NaPy concentration in the growth media and in the pre‐treatment. We analysed each cell type separately using a two‐factor ANOVA with replication for the different conditions of growth media and pre‐treatment media. Then, we performed a Tukey post‐hoc multi‐comparisons of the group means to compare the percentage change in dextran uptake across the experimental conditions. A P‐value of 0.05 was set as the significance level of the multiple comparison test.

3. RESULTS

By applying damaging‐level strains to murine fibroblasts (NIH3T3) and myoblasts (C2C12), cell types typically found at susceptible superficial and deep tissue sites,22 we model initial damage to cell structure at those sites (Figure 1); if not prevented, the damage may cascade to PU, whereas these motile cell types would facilitate closure of gaps.20 We evaluate the effects of varying concentrations of the NaPy additive on the cells' ability to maintain the integrity of their PMs under damaging‐level strains. The NaPy is provided in the cell growth media (ie, prolonged exposure) and is also added as a pre‐treatment 4 hours prior to damage induction (Figure 3). Damage is induced in the cells by sustained, radially applied 12% strains for 3 hours, and changes to membrane integrity are quantified by the uptake of a small marker molecule as compared with unstrained control.

Figure 4 demonstrates the dextran uptake following cell damaging‐level (12%) strain loading under different NaPy pre‐treatment concentrations (0, 1, or 5 mM) in a single, representative experiment with the C2C12 cells grown in NaPy‐free media. The fluorescence intensity histograms provide the background AF (left two curves) and the fluorescent‐dextran internalisation (right two curves), both following 3 hours of damaging (12%) control (0%) stretching (strain loading). The AF of the strained and unstrained samples was similar in each experiment, regardless of NaPy pre‐treatment concentration; the absolute values differ between experiments. The natural uptake of the small dextran is not significantly affected by NaPy pre‐treatment concentrations. Strain‐induced dextran uptake into cells is indicated by a right shift of the curves. Specifically, when no NaPy pre‐treatment was added prior to straining (0 mM NaPy in Figure 3), we observe increased uptake of dextran (Figure 4A, right shift of curve). The strain‐induced marker uptake is reduced with increasing NaPy pre‐treatment concentration (left shift of the curves in Figure 4B,C), bringing the uptake closer to that of the unstrained cells; a similar response is observed in both cell types. To more generally evaluate the effects of cell type, NaPy concentration in growth media, and NaPy pre‐treatment concentration on changes in PM permeability following damaging‐level strain loading, we vary conditions and determine the average change in uptake.

Figure 4.

Representative experiment of fluorescent distribution curves obtained by fluorescence‐activated cell sorting (FACS) experiment with C2C12 myoblasts cultured in growth media with 0 mM sodium pyruvate (NaPy) (NaPy free) prior to an experiment. The effect on fluorescent dextran uptake, that is, plasma membrane permeability is shown for pre‐treatment with (A) 0, (B) 1 and (C) 5 mM NaPy. The curves correspond to the strained (orange shades) and unstrained (blues) conditions; similar plots were obtained for the NIH3T3. The dashed line highlights that the auto‐fluorescence (without dextran) of the strained and unstrained cells is indistinguishable regardless of NaPy pre‐treatment, which we have observed consistently in both cell types and under both growth media NaPy concentrations (0 or 1 mM)

By quantifying changes in the uptake of the fluorescent‐dextran marker, we observe increased membrane poration under damaging‐levels strains (12%), which is reduced by NaPy pre‐treatment in a concentration‐proportional manner (Figure 5); change in uptake is calculated relative to the unstrained baseline while subtracting the respective background AF (see Section 2). To prevent the evaluation of initial cell damage that may be repaired by the cells (eg, utilising the excess, available NaPy), we have only added the FITC‐dextran marker to the cells during the last 30 minutes of the strain damage infliction (Figure 3). We observe that the pre‐treatment of myoblasts (Figure 5A) and fibroblasts (Figure 5B) with increasing NaPy concentrations proportionally reduces the strain damage‐induced PM poration and permeability, which are precursors to loss of cell homeostasis and death (Figure 1); differences in dextran uptake with increased NaPy concentration indicated in Figure 5 were statistically significant (P < .03). We note that, in both cell types, the largest relative increase in PM permeability because of damaging strains (relative to unstrained control) occurs when no NaPy pre‐treatment is applied. However, long‐term exposure of both cells types to NaPy in the growth media (1 mM), without pre‐treatment immediately prior to damage induction (0 mM on x‐axis of Figure 5), significantly reduces (P < .04) the strain‐induced membrane poration. Concurrently, in the myoblasts, long‐term exposure to NaPy in the growth media combined with NaPy pre‐treatment increases the cell‐protective effect, bringing the 12% strained cells closer to the baseline uptake observed in the unstrained controls. Specifically, for myoblasts under a treatment protocol that includes long‐term exposure to 1 mM NaPy in the growth media and then pre‐treatment with 5 mM NaPy prior to application or damaging strains, the internalisation or membrane poration becomes indistinguishable from the unstrained (undamaged) control (Figure 5A). In the NIH3T3 fibroblasts, in contrast, when long‐term exposure to the NaPy is not provided, short‐term exposure is sufficient to significantly (P < .03) reduce the membrane poration under damaging‐level (12%) strains (Figure 5B). Hence, it is the lasting effects of NaPy during the strain application that reduces the damage‐induced poration in the cells' PMs.

4. DISCUSSION

Our results show that prolonged exposure and pre‐treatment with exogenous NaPy supplement, respectively, days and hours prior to the initiation of damaging‐level mechanical loading reduce cell PM poration in a statistically significant manner, with a concentration‐dependent effect. PM poration may lead to the loss of homeostasis and the eventual death of cells, which is the micro‐scale, initial stage of PU formation (Figure 1). We observe that the exogenous NaPy supplement allows cells to maintain the (mechanostructural) integrity of their cytoskeletons and the PMs1 when applied prior to damage induction. We have demonstrated this in both myoblasts and fibroblasts that are simple in vitro models of the cell types prevalent at, respectively, susceptible superficial and deep tissue sites; those are likely to be involved in the PU formation process, as well as in early‐stage repair of local damage.

In the current study, cell strain loading is induced by the application of sustained, damaging‐level (12%) strains to cell monolayers. Under sustained strains, it is not unreasonable to expect cell relaxation or adaptation over time. We have, however, observed indications of long‐term effects because of sustained strains, even with smaller non‐damaging strains that have been applied for longer time scales. In recent work, we had observed that, in terms of migration rates of fibroblasts or of myoblasts, extended exposure to NaPy (in growth media and prior to damage) was only sufficient to accelerate gap closure if low‐/mid‐level (3%‐6%) sustained strains were added or combined with post‐injury exogenous NaPy supplement.20 The gap closure process was on the scale of 24 hours, indicating the long‐term effects of the sustained stretching strains.

We note that, in fibroblasts, using a simple model of a prevalent skin constituent, cell protection by NaPy may be attained (preferentially) by pre‐treatment applied a few hours prior to damage induction or also by long‐term (days) exposure (Figure 5B); combining the exposure routes does not significantly improve the protective effect. Micro‐scale gaps in tissue (resulting from the death of hundreds of cells) may still form under prolonged or excessive loading or if cells are no longer able to adaptively respond, for example, if exogenous NaPy supplement has been depleted.1 In that case, we have recently shown that pre‐exposure to NaPy would accelerate gap closure by cell migration (repair), even if the exogenous pyruvate reservoir had already been depleted, as long as compensatory low‐level, non‐damaging, sustained strains are applied as a combined treatment.20 This is especially critical for patients whose skin is more fragile, leading to the rapid formation of PUs on the skin, for example, because of contact with medical devices of various types, utilised in life‐saving procedures that inadvertently cause damaging‐level mechanical loading on skin and deeper tissues.6, 7 For those patients, one can envision preventative pre‐treatment with NaPy to avert PU formation at susceptible sites on the skin by facilitating the fibroblasts' dynamic adaptation to avoid damage concurrently by accelerating their ability to repair small‐scale damage in the form of micro‐scale gaps (resulting from the death of hundreds of cells) before the development of a PU.

The preventative effect of NaPy is even more pronounced on C2C12 myoblasts, which are relevant for initiation of DTIs; myoblasts are undifferentiated muscle cells and are highly migratory, allowing them to repair muscle tissue damage. We observe that pre‐treatment with NaPy for hours prior to damage induction facilitates protection of the myoblast cells from mechanostructural damage in a dose‐dependent manner (Figure 5A). Combining long‐term treatment, for example, prolonged exposure to 1 mM NaPy (in the growth media), significantly improved the protective capacity of NaPy, maintaining the dose dependence yet facilitating reduction of the damage to control (unstrained) levels. Chronic, 72‐hour treatment of C2C12 myoblasts with supra‐physiological concentrations of pyruvate (50 mM) has previously been shown to induce mitochondrial biogenesis by an increase in mitochondrial mass and functionality.56 Thus, in deep skeletal muscles, prolonged exposure to NaPy may have cumulative positive effects over time; this would also have protective relevance to longer time‐scale phenomena such as ischaemia. Aptly, we have recently shown that, in myoblasts with extended exposure to exogenous NaPy supplement (1 mM in growth media), a combined treatment of NaPy supplement and low‐level (3%–6%) non‐damaging, sustained strains applied together, immediately following cell damage/death infliction, had significantly accelerated closure rates of micro‐scale gaps.20 Specifically, in the C2C12 myoblasts, grown in 1 mM exogenous NaPy, an in vitro “sweet spot” was identified in terms of gap closure rate acceleration: combination of 3% sustained stretching strain with 5 mM NaPy post‐injury. Consistently, in terms of maintaining PM integrity, we observe that prolonged pre‐exposure combined with pre‐treatment immediately before damage induction significantly increases the protective NaPy effect, reducing poration of the myoblasts to that of the controls. Thus, the exogenous NaPy pre‐treatment accelerates gap closure and, as shown here, also allows cells to avert mechanostructural damage, that is, loss of PM integrity, which can lead to the loss of homeostasis and cell death. The preventative effects of NaPy, especially when combined with non‐damaging, low‐level strains, can delay the onset and development of PUs, thus extending the safe time of immobile persons in a position.

Exogenous pyruvate provides many metabolic and energetic advantages to cells, neutralises reactive oxygen species (ROS), and reduces their generation in the mitochondria while also maintaining PM potential during oxidative stress. Anti‐inflammatory effects are irrelevant during the early stages of PU initiation (ie, prior to involvement of the body's inflammatory system13), which are tested in the in vitro systems used here. Pyruvate acts as a substrate for adenosine triphosphate (ATP) production in the mitochondria during the Krebs cycle.34 in vitro studies have shown that pyruvate may reduce damage in normal and cancer cells by acting as an antioxidant,37, 48 as well as an efficient energy source.57, 58 The available energy facilitates a dynamic response of the cytoskeleton26 as is also explicitly observed in highly dynamic and energetic cancer cells.28, 29 Thus, cell protection from damaging mechanical loads is likely enabled by the increased available energy and the antioxidant capacity afforded by the exogenous NaPy supplement. NaPy may reduce cell damage by acting as an efficient energy source,48, 58 maintaining local energy reserves.59

Our results indicate that pre‐treatment with NaPy prior to strain loading (ie, expected periods of immobility in vivo) can reduce PM poration, likely by facilitating rapid, adaptive cytoskeletal responses to redistribute cell loads1 while scavenging any ROS. Similarly, antioxidative scavenging of ROS by pyruvic acid maintained the viability of Osteosarcoma 143B cells under aggressive oxidative stress (1 mM H₂O₂) by 30 minutes of pre‐incubation with 5 mM pyruvate.60 We note that prolonged exposure to NaPy can improve its protective capacity, potentially extending the protective time and thus extending the safe time in a position. The antioxidant capacity of NaPy is specifically relevant for treatment of ischaemia, which typically appears at later stages of PU development.13, 21 Pyruvates have been shown to protect fibroblasts and embryonic stem cells following prolonged (72 hours) oxidative stress,61 to minimise deficits after traumatic myocardial infraction in pigs,41, 42 and to reduce cortical contusion injury and hypoxic‐ischaemic damage in rat brains.43, 44, 45 All these correspond to the longer time scale supportive advantages of pyruvates. However, treatments have been shown to be most effective if applied within the first 2 to 6 hours of damage43; otherwise, the benefits are only transient.62 Hence, NaPy supplement, if added before initiation of damage or close to it, has potentially positive short‐term and long‐term effects, well beyond time scales of the initial mechanical loading‐induced damage to cells and tissues.

The effects of the exogenous NaPy supplement applied both as long‐term treatment (eg, here in the in vitro growth media) and in the pre‐treatment before expected periods of immobility, where damaging loads are expected, can be developed into clinical technologies (Patent Pending,27 co‐inventor DW). As pyruvates also generally provide beneficial antioxidant and anti‐inflammatory activities, any exogenous NaPy supplement remaining in the tissues after preventing initial loading damage (eg, averting cell death or closing micro‐scale gaps) could then be used to provide longer‐term treatment for residual effects, such as remaining ROS or secondary inflammatory damage, for example, related to ischaemic damage.13 This preventative technology can be designed to be delivered, for example, through the skin by various approaches, such as through prophylactic dressings, garments, or even bedding.27 The preventative approach is important and relevant for all susceptible populations, including persons with temporary or permanent sensory impairment, such as persons undergoing surgery, anaesthesia, or are otherwise immobilised. Prophylactic pre‐exposure to NaPy could also potentially provide protection against PUs that may result from loads applied to the skin, for example, by medical devices. In such cases, an exogenous pyruvate supplement can provide preventative protection against the initiation of PUs under damaging loads by allowing cells to dynamically respond and avoid mechanostructural damage that can lead to cell death.

CONFLICT OF INTEREST

No competing financial interests exist. The content of this article was expressly written by the authors listed. No ghostwriters were used to write this article.

Alvarez‐Elizondo MB, Barenholz‐Cohen T, Weihs D. Sodium pyruvate pre‐treatment prevents cell death due to localised, damaging mechanical strains in the context of pressure ulcers. Int Wound J. 2019;16:1153–1163. 10.1111/iwj.13173