Study design. The study was designed to compare the concentrations of cytokines (TNF-α, IL-1β, IL-10) and growth factors (IGF-1, TGF-β1) in blood serum and their correlations with changes in the surface area of stage 2 through stage 4 PIs after 4 weeks of treatment among 3 parallel groups of patients receiving SWC plus anodal HVMPC (AG), SWC plus cathodal HVMPC (CG), and SWC plus sham HVMPC (PG), respectively.
The results of this study are part of a prospective, randomized controlled clinical trial designed to gain insight into the effects of anodal and cathodal electrical currents on the PI healing process. The authors’ previous study31 examined the clinical effects of healing stage 2 through stage 4 PIs and changes in blood flow at the edge of PI after treatment with anodal, cathodal, and sham ES among 61 patients. In the current study, changes in blood levels of cytokines and growth factors were assessed in 43 patients, 40 of whom (65.57%) were part of the previous research31 and had agreed to donate blood samples. The study was conducted from December 1, 2015, to January 30, 2017; 3 additional patients were included in the more recent study from January 30, 2017, to February 27, 2017.
Study enrollment and criteria. Patients screened for the study were treated as inpatients at one rehabilitation center between December 1, 2015, and February 27, 2017. Their eligibility to participate was assessed by their physician using the following main criteria: neurological injuries (spinal cord injury [SCI], ischemic stroke, blunt trauma to the head), 18+ years old, at high risk of PI development (>15 points on the Waterlow scale),46 and having a stage 2, stage 3, or stage 4 PI of at least 0.5 cm2 in size and at least 4 weeks’ duration, located on the pelvic girdle or lower extremities.
An additional criterion for inclusion in the study was consent to donating blood samples. Patients with local ES contraindications (cancer, electronic implants, osteomyelitis, tunneling, necrotic wounds) were excluded from the study, as well as patients with PI requiring surgical intervention, poorly controlled diabetes mellitus (HbA1c >7%), critical wound infection, allergies to standard wound treatment, and/or no consent for donating blood samples. Patients were informed in writing by the research manager about the aim and course of the study and that they could withdraw from the study at any time without any consequences on their further treatment.
Randomization. Patients were randomly allocated to 3 groups using sealed envelopes containing special codes (A for the AG, B for the CG, and C for PG). The letters were inserted into envelopes that were sealed and then numbered randomly in computer-generated order. The envelopes were delivered to the main investigator, who opened them and directed the patient to the appropriate group according to the enclosed symbol.
Blinding. All patients, medical personnel, and researchers were blinded as to what type of ES was being applied to patients (anodal, cathodal, or sham ES). The exceptions were the main investigator and the principal physiotherapist who set the ES device to apply active or sham ES. The persons measuring the area of the wound surface, determining the levels of cytokines and growth factors in the blood, and performing statistical analysis of the results of the study also were blinded.
Study variables. Demographic information on the patients enrolled in the study was obtained from standardized participant interviews, physical examinations, the results of additional examination, and history of concomitant diseases. Study variables for assessing wounds, PI risk, and patients’ nutritional status were collected with paper-and-pencil instruments and transferred to a data sheet. Patient case history, results of blood laboratory tests, and previous diagnostic and treatment results were obtained from the electronic hospital database. All obtained information then was entered to a computer database to facilitate analysis.
Standard wound care administered to all groups. All patients in the AG, CG, and PG received SWC (scheduled repositioning, wound dressing changes, and physiotherapy treatment) under the supervision of the physician and the principle investigator and following best practices.14,47,48 To protect the trial participants from developing more PIs, pressure-redistribution surfaces, foam devices, and pillows were provided. Patients who were immobile were repositioned by a nurse or physiotherapist at least every 2 hours, and persons who could move were instructed to change position as often as they could.
Patients who were diagnosed with malnutrition based on weight observation and blood tests received individual nutritional support.14 Patients’ wounds were examined regularly by a physician to determine the appropriate type of topical treatments. Tissue debridement was combined with infection and inflammation control, maintaining moisture balance in the wounds, and monitoring epithelial wound edges and epithelialization. Before ES was applied, necrotic tissue was removed from PIs with surgical/sharp, conservative sharp, or enzymatic debridement. Patients with elevated leukocyte levels received antibiotics selected according to the results of microbiological culture and sensitivity tests. All immobile patients received low-molecular-weight heparin. SWC, nutritional support, and general treatment of patients have been described in detail in the authors’ previous publication.31
Anode group. Patients in the AG were administered SWC and anodal HVMPC energy. The device used to deliver HVMPC was the Intelect Advanced Combo (DJO Global, Vista, CA); the device has 2 independent electrical circuits, but only one was active. The device generated a twin-peak monophasic pulse (154 µs) consisting of 2 77-µs exponential pulses in rapid succession. Pulse frequency was set at 100 pps and voltage above 100 V for amperage of 0.36 so as not to elicit motor reactions. The electrodes delivered a charge of 360 µC per second and 1.08 C per day. Patients participated in 50-minute sessions every week day (Monday through Friday). All patients had a personal set of conductive carbon-rubber electrodes. During the procedure, the treatment electrode (50 cm2) was placed on the wound and the return electrode (100 cm2) was applied to healthy periwound skin at least 20 cm from the PI. Both electrodes were separated from the tissue by aseptic gauze pads saturated with physiological saline. Anodal stimulation was applied to PIs once a day.
Cathode group. HVMPC protocol in the CG was identical, except cathodal instead of anodal stimulation was used.
Sham group. This group received SWC and sham HVMPC. The arrangement of electrodes during the procedure was the same as in the ES groups. The monitor of the ES unit displayed all parameters, but because the electrodes were connected to the inactive electrical circuit, current energy was not delivered to wounds.
General protocol. For all treatments, the lead physiotherapist connected the electrodes and selected the polarity of the treatment electrode. The procedure was performed in an inconspicuous manner so neither the patient nor the members of the medical team could see whether real or sham ES was applied.
In the active ES groups, amperage was set to 0.36 A (the same value was displayed on the monitor for patients receiving sham ES), which did not cause muscle contractions but only weak tactile sensations. Because most patients in the groups had tactile sensory problems and did not perceive the current, patients in the sham ES group did not know that they were not being treated. All treatment sessions had the same duration and frequency and followed the same protocol whether sham or active ES was applied.
The electrodes were sterilized before and after each session in approved disinfectant solution. As soon as the procedure ended, patients’ wounds were washed thoroughly with a 0.9% sodium chloride solution and covered with SWC dressings. In patients with more than 1 PI, all wounds were treated, but only the most severe were analyzed statistically.
Cytokine and growth factor concentrations. To determine cytokine and growth factor concentration in blood serum, peripheral blood samples (5 mL) were taken from the patients twice, immediately before treatment and at week 4. Blood serum was centrifuged away and stored at -80˚ C until the levels of cytokines and growth factors were determined. TNF-α, IL-1β, IL-10, and TGF-β1 levels were assessed using the immunoenzyme method (ELISA) using the following kits: Human TNF-α ELISA kit (Diaclone, Besancon Cedex, France), Human IL-1β ELISA kit (Diaclone), Human IL-10 ELISA kit (Diaclone), and TGF-β1 kit (Cloud-Clone Corp, Katy, TX). The concentration of IGF-1 was measured by chemiluminescence and a set of Human IGF-1 kit reagents (DiaSorin, Saluggia, Italy).
Wound measurements. Wound surface area (WSA, calculated in cm2) measurements were taken at baseline and after each week of therapy. The WSA was determined by tracing wound shapes onto acetate sheets and from the sheets onto rigid, transparent film for measurement with a planimeter. Measurements were processed by a digitizer (Mutoh Kurta XGT, Altek, Phoenix, AZ) connected to a personal computer with C-GEO software (version 4.0, Nadowski SoftLine, PL) that also was used for making computations and storing the results.
Primary outcomes. The primary outcomes of the study included:
1. Percentage changes in blood serum concentration of pro-inflammatory cytokines (TNF-α and IL-1β) at week 4 of treatment compared with pretreatment levels;
2. Percentage changes in blood serum concentration of anti-inflammatory cytokine IL-10 at week 4 of treatment compared with pretreatment levels;
3. The percentage change in the concentration ratio of pro-inflammatory cytokine IL-10 to inflammatory cytokine TNF-α (IL-10/TNF) in blood serum at treatment week 4 compared with pretreatment values; and
4. The percentage change in the concentration of growth factors IGF-1 and TGF-β1 in blood serum at treatment week 4 compared with pretreatment values.
The formulas for calculating the percentage changes listed above are presented in Table 1.
Secondary outcomes. Secondary outcomes included correlations between the concentrations of cytokines and growth factors in blood serum and the decrease in the PI size.
Intention-to-treat analysis. To retain data of all randomly allocated participants, an intention-to-treat analysis was performed. Data that were not available were approximated using an exponential regression function written as WSA = b exp(-at), where WSA is wound surface area; b and a are the regression constant and the exponential regression coefficient calculated for each patient using WSA (cm2) obtained over the period of treatment, respectively; exp is the exponential regression function with a base of e≈2.718282 (the Euler’s number); and t is the week of treatment.49 The function allows WSA decreases to be described and can be calculated with data from at least 3 weeks.49 The exponential correlation coefficient proved negative for each patient and higher than 0.9 for the absolute WSA.
To minimize the risk of baseline interpatient differences biasing the results of the study, the relative values were used to determine the changes of cytokine and growth factor concentration in blood serum at week 4 and to calculate correlations between cytokine and growth factor concentration in blood serum and percentage reductions in wound surface area.
Patient characteristics were tested for normal distribution using the Shapiro-Wilk W-test, which showed that their distribution was not normal. Levene's test revealed heterogeneity of variance. Despite the absence of normal distribution, because of low absolute values of skewness and kurtosis (<2.5), a mean was used as the central value and a standard deviation as a measure of dispersion.
The baseline homogeneity of patient characteristics between groups was assessed using the maximum-likelihood chi-squared test, the analysis of variance (ANOVA) Kruskal-Wallis test, and the Kruskal-Wallis post-hoc test. To compare mean percent changes in blood levels of cytokines and growth factors between groups, the ANOVA/ Kruskal-Wallis test and Kruskal-Wallis post-hoc test were employed.
The correlations of cytokine and growth factor with percentage wound area reduction (PAR) were tested with Spearman’s rank order test. The following correlations were calculated:
1. Between percentage changes of cytokines (TNF-α, IL-1β and IL-10) at week 4 and the PAR noted at week 4 (%PAR 4) and at week 8 (% PAR 8) (see Table 2); and
2. Between percentage changes of growth factors (IGF-1 and TGF-1β) at week 4 and the PAR noted at week 4 and at week 8.
In all tests, the level of significance was P <.05. All statistical procedures were blinded and utilized Statistica software, version 13.1 (StatSoft Polska Sp. z o.o.).
Ethical approval. Ethical approval was granted by the Academy Bioethics Commission.
The trial was registered with the Australian-New Zealand Clinical Trials Registry: ACTRN12616001709437; and funded by the J. Kukuczka Academy of Physical Education (Katowice, Poland).