Comparison of a Saline-coupled Bipolar Sealer Versus Traditional Electrosurgery in a Porcine Model of Chronic Wound Healing

Objective. This study examines the healing dynamics of in vivo porcine muscle tissue wounds hemostatically treated with a saline-coupled bipolar tissue sealer (SCBS) compared with traditional electrosurgical (ES) coagulation. Materials and Methods. Six cutaneous incisions were created on the dorsum of 28 adult male Yorkshire swine. The underlying muscle tissue was incised with a cold scalpel then treated with either SCBS (at 170 W) or traditional ES (at Coag 45 W). Time to hemostasis was recorded. Animals were humanely euthanized at day 2 and weeks 2, 3, or 8; treated tissue was harvested for histopathological evaluation. Results. After 8 weeks, the extent of wound healing was similar between SCBS and ES. Both devices controlled bleeding effectively; however, SCBS-treated wounds exhibited a greater depth of thermal effect over the first 3 weeks despite a shorter treatment time. Wounds treated with SCBS demonstrated fewer inflammatory markers at early time points but healed more slowly, with scores that lagged behind ES for collagen deposition, fibrous tissue maturity, extracellular matrix, and stage of healing. Myofiber regeneration notably increased in SCBS-treated wounds at weeks 2, 3, and 8. By the end of the 8-week recovery period, there were no significant differences in healing parameters between the 2 groups. Conclusions. Overall, both devices elicited similar progression of healing by 8 weeks. The SCBS produced a deeper thermal effect in a shorter treatment time and improved myofiber regeneration compared with ES and had an equivalent overall course of healing. 

Electrosurgery (ES) — the use of a monopolar, radiofrequency-energized electrode for soft-tissue dissection, coagulation, and hemostasis — was introduced as a surgical tool in the late 1920s.¹ Since then, ES has become a standard instrument in the operating room, used as a mainstay in a wide range of inpatient and outpatient surgical procedures. Although its hemostatic and dissection capabilities were major advances that remain indispensable to surgeons today, ES is associated with a number of undesirable effects: thermal injury to collateral tissues, increased inflammation, and delayed wound healing compared with cold scalpel incisions.²

Much of the negative effect that traditional ES exerts on wound healing may be due to its fundamental operating design, which has not changed significantly since it was first developed.^3-5 While traditional devices vary in some features, the underlying principle is the same: radiofrequency current created by a generator passes through a bare, uninsulated metal electrode (blade) to the patient at the point of surgical contact and then through the patient to a grounding (return) pad that completes the circuit. During dissection using Cut mode, tissue is vaporized by the rapid superheating of intracellular liquid by the application of high electrical current; conversely, hemostasis using the Coag mode is achieved through thermal denaturation and coagulation of tissue by the application of high voltage. In both applications, a standard ES electrode can reach operating temperatures between 250°C and 350°C. Subsequent thermal stacking and heat diffusion are the principal sources of thermal tissue damage.²

A number of innovative alternative device designs have been introduced to the market to address the problems of high electrode operating temperatures and thermal damage to tissue during dissection and hemostasis. These include the PEAK PlasmaBlade (Medtronic, Minneapolis, MN), which features a highly insulated electrode and a pulsed radio frequency (RF) waveform that reduce electrode operating temperature; the Aquamantys saline-coupled bipolar tissue sealer (SCBS; Medtronic), which couples bipolar RF energy with saline cooling; and the Harmonic Scalpel (Ethicon, Somerville, NJ), which uses ultrasonic vibration to effect cutting and coagulation.^2,6-8 

In vivo studies have been conducted comparing the effects of low-thermal injury dissection devices and traditional ES on healing response after dissection. These studies examined microscopic histological parameters, such as depth of thermal damage, inflammatory cell infiltration, smooth muscle actin and collagen deposition, wound organization, and macroscopic parameters, such as cosmesis and healed wound strength.^2,7,9–11 Device manufacturers and independent groups also have published studies assessing healing response versus ES following the use of ferromagnetic wands,¹² ultrasonic dissection devices,^8,13–15 and laser-assisted technologies.^12,16

The SCBS systems are used principally for hemostatically sealing of soft tissue and bone at the operative site, primarily in open orthopedic procedures such as total hip arthroplasty, total knee arthroplasty, and spinal fusion. While a number of studies have been published describing the hemostatic capabilities of SCBS,^17–19 to the best of the present investigators’ knowledge, there has not yet been a rigorous evaluation of the effects of SCBS hemostatic treatment on chronic wound healing compared with ES. 

Study overview
This was an open-label, non-Good Laboratory Practice, prospective, randomized, controlled, head-to-head comparison of chronic wound healing in a live porcine model. The objectives of the study were to characterize and compare chronic wound healing after hemostatic treatment of skeletal muscle incisions by the SCBS and ES devices, as evaluated by observing fibrovascular response (granulation tissue), remodeling, and regeneration.

The primary objective was to assess and characterize overall wound healing by obtaining semiquantitative histological data at defined time points. The secondary objective was to obtain quantitative data on depth of thermal injury, microvessel density, edema, and neutrophil, lymphocyte, monocyte, and macrophage infiltration.

Animals
All study protocols were reviewed and approved by the Medtronic Institutional Animal Care and Use Committee and conducted in accordance with US and international regulations for protection of laboratory animals. Twenty-eight adult male Yorkshire swine (60 kg–70 kg; age, 6–7 months) were housed and fed according to Medtronic Physiologic Research Laboratory protocol and were acclimatized prior to all study procedures.

Devices
The SCBS device was the Aquamantys 6.0 (Medtronic Advanced Energy, Portsmouth, NH), a sterile, single-use, hand-activated, bipolar electrosurgical device powered by the AQM Pump Generator which delivers RF energy and saline for hemostatic sealing and coagulation of soft tissue and bone at the operative site. 

The ES device was the Valleylab electrosurgical pencil (model E2450H; Medtronic) with the Edge PTFE (Telfon)-coated electrode (model E1450X), a sterile, single-use, monopolar electrosurgical device powered by the Valleylab Force FX-C (Medtronic) generator.

Study surgical procedure
For each animal, anesthesia was induced with isoflurane, and general anesthesia was administered with sevoflurane via endotracheal intubation. Paralysis was induced with succinylcholine choline 40 mg/kg intravenously to prevent muscle twitching during treatment. 

Using ES, a single user prepared six, 7-cm cutaneous incisions on the dorsum of each swine, parallel to the spine, to expose the underlying skeletal muscle (Figure 1). These treatment areas were arranged symmetrically and bilaterally, equidistant from the dorsal spinous process and at least 2 cm apart laterally. 

Next, a cold scalpel was used to create 1-inch long incisions in the exposed muscle tissue of each treatment area to a depth of 0.5 inch. Following incision, treatment was applied to the raw edges of each incision using SCBS, ES, or the control treatment according to a predetermined randomization scheme. Generator power settings were selected according to standard clinical practice in orthopedic procedures (SCBS at 170 W with medium saline flow; ES at a setting of Coag 45 W). Treatment was applied in a manner suitable to effectively treat a 1-in² area in a uniform fashion. Effective treatment was defined as a single pass in a painting motion until tissue effect (hemostasis and color change) was achieved. Application time (time required to achieve hemostasis) was recorded for each experiment. The control treatment consisted of creating cutaneous and skeletal muscle incisions with a cold scalpel, with no hemostatic treatment. Following hemostasis, each wound was dried with gauze and closed in layers with continuous (subcutaneous) and interrupted (cutaneous) 3-0 nylon sutures.

All animals underwent the surgical procedure on the same day (day 0) and were allowed to recover from surgery. Either SCBS or ES treatment was applied to at least 1 of the 6 wounds on every swine, and the control treatment was applied to a minimum of 1 wound on at least 6 of the 7 swine euthanized at each time point.

Sample collection and histological analysis
Animals were humanely euthanized at the end of their specified recovery period (2 days or 2, 3, or 8 weeks after treatment). Each treated section of muscle was excised to yield a minimum of 3 cross sections through the treatment area, fixed in 10% neutral buffered formalin, paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E) for histology processing. A single pathologist blinded to the treatment modality evaluated each specimen under confocal light microscopy for inflammatory cell infiltration, collagen formation, fibroblast proliferation, angiogenesis, and granulation-tissue formation. Immunofluorescence staining with monoclonal antibodies (PerkinElmer, Inc, Hopkinton, MA) specific to smooth muscle actin (to detect collagen regeneration) and cluster of differentiation 31 (CD31) (to detect angiogenesis activity) was used to identify and quantify the amount of protein present in tissue sections.

The pathologist assessed microscopic evidence of wound healing according to the objective and semiquantitative scoring system (eTable 1). The scoring criteria take into account the degree of inflammatory response, amount and organization of collagen deposition, fibrous tissue maturity, microvessel density, and stage of healing on a scale of 0 to 3.  A lower score denotes better outcomes. 

Quantitative microscopic evaluation of the depth of thermal injury also was performed, including measurement of the maximum width of the zone of coagulation necrosis by H&E staining and of microvessel density, edema, neutrophil, lymphocyte, monocyte, and macrophage infiltration by counting the number (or percentage) of relevant cells per high-powered field (HPF; eFigure 2). 

Statistics
The animal model to assess chronic healing was adapted from 2 previously published wound healing studies,^7,20 and sample size was calculated using a t statistic. Assuming a 10% failure rate, with a standard deviation of 50% for a power of 90% with α = .05, the minimum sample size (number of treatments per device, per time period) was calculated to be 12. Twenty-eight total animals (168 treatments) were used to provide adequate statistical power while accounting for any attrition due to death. This provided for 18 treatments per device, plus 6 control treatments, per time period. 

The null hypothesis (H0) was that wound healing, tissue reperfusion, and inflammation in areas treated with the SCBS would be equivalent to those in areas treated with ES at 2 days and 2, 3, and 8 weeks following the procedure. 

All quantified values are presented as mean ± standard deviation. Comparisons between groups were assessed using analysis of variance, followed by t test or Kruskal-Wallis H test for statistical significance, with a prespecified alpha of .05. Error bars were plotted as 95% confidence intervals (CIs).

All scheduled procedures were carried out as planned, and all animals survived with no remarkable morbidity until euthanasia.  A total of 168 samples were analyzed (72 SCBS, 72 ES, and 24 control).

Histological comparison of characteristics of acute injury and inflammatory response are shown in eFigures 2, 3, and eTable 2. Average scores for each parameter are presented on a scale of 0 to 3 (eTable 1 has specific scoring criteria), with lower scores signifying better outcomes.

As expected, edema was most pronounced (highest scores) at the 2-day time point in all samples and declined over the 8-week period of wound healing (eFigure 3A). There were no significant differences at any time point in level of interstitial edema between wounds treated with SCBS and ES. 

Hemorrhage was mild or absent, with mean scores less than 1 for all time points in all samples and no significant differences between groups (eFigure 3B). Semiquantitative scores for depth of thermal injury to myofibers (eFigure 3C) were significantly higher for SCBS versus ES at 2 days (2.42 ± 0.19 vs. 1.60 ± 0.19), 2 weeks (2.31 ± 0.21 vs. 1.87 ± 0.19), and 3 weeks (1.87 ± 0.19 vs. 0.07 ± 0.19), with P < .001.

The density of specific cell types associated with healing was scored on a 0 to 3 scale (eTables 1, 2). At the early time points, mean scores for overall infiltration of inflammatory cells (mean cell count per HPF) were higher for ES-treated wounds than SCBS (2.89 ± 0.18 vs. 2.37 ± 0.18 at 2 days, respectively, and 2.56 ± 0.18 vs. 2.15 ± 0.20 at 2 weeks, respectively; P < .001), but scores were higher for SCBS-treated wounds at 3 weeks (2.23 vs. 1.5). By 8 weeks, cell infiltration was scored as 0 (< 1–10 cells/HPF) for both groups. The density of neutrophils, the presence of which is associated with the acute inflammatory stage of wound healing, was significantly higher in ES-treated wounds compared with SCBS at day 2 and week 2, and it was low or not present at weeks 3 and 8 for both groups. Differences in lymphocyte density were only significant at the 2-week time point, with ES scoring higher than SCBS. Scores of macrophage density were significantly higher in SCBS compared with ES at day 2 and week 2. No difference was seen at week 3, and the 8-week scores were 0 for both groups. There were no significant differences in the presence of multinucleated cells at any time point, and no plasma cells were observed at any time point.

Histological characteristics associated with the stages of wound healing and tissue remodeling are presented in eFigure 4. As expected, collagen was absent in samples from both groups at day 2, with increasing evidence of deposition and maturation over the 8-week course (Figure 4A). As evidenced by lower scores at the 2-week and 3-week time points, collagen deposition and maturation appeared to progress more quickly in wounds treated with ES compared with SCBS, but, by 8 weeks, both groups scored less than 1 with no significant differences. Similar patterns were seen for fibrous tissue maturity (eFigure 4B) and appearance of extracellular matrix (eFigure 4C). Microvessels were not observed in either group at day 2, and microvessel density improved over time; no significant differences between groups were noted (eFigure 4D). 

eFigure 5 shows an overall assessment of the stage of healing. As expected, little to no healing was observed in samples from either group at day 2 (score of 3), with stepwise progression until healing was considered good/complete at the 8-week time point. Consistent with characteristics of tissue structure in eFigure 4, ES-treated samples scored significantly lower compared with SCBS at weeks 2 and 3, but by 8 weeks there were no differences observed in healing stage between wounds treated with ES and SCBS.  

An immunohistochemical assessment of inflammation, smooth muscle actin, and angiogenesis (CD31) yielded minimal evidence of differences between the SCBS, ES, or control groups (eTable 3). 

The time required to treat the wound area for SCBS and ES was recorded (ie, time to tissue effect as evidenced by hemostasis and color change). Treatment time was significantly shorter for SCBS-treated wounds compared with ES (6.8 ± 0.57 seconds; 95% CI, 0.13 vs. 18.8 ± 3.3 seconds; 95% CI, 0.78; P < .05; eFigure 6). 

Wound healing is a complex, orderly phenomenon involving a number of sequential, overlapping processes, including induction of the acute inflammatory process, regeneration of parenchymal cells, migration and proliferation of both parenchymal and connective tissue cells, synthesis of extracellular matrix, tissue remodeling, and collagenization. The acute inflammatory phase, which lasts for a few days, is characterized by edema and leukocyte (predominantly neutrophil) infiltration. Subsequent chronic inflammation features the initiation of tissue repair cascades and the release of cytokines involved in granulation, collagen remodeling, and angiogenesis, and may last for months. These events ultimately lead to the maturation of fibroblasts into myofibroblasts expressing α-smooth muscle actin, with the resulting tissue repair and scar matrix maturation. 

Published clinical studies have demonstrated the utility of the SCBS system for hemostasis in a variety of procedures, including total hip arthroplasty,^18,21 spinal surgery,²² brain tumor surgery,²³ hepatectomy,⁶ and knee arthorplasty.^17,19 While these have imparted useful information regarding patient outcomes, the device’s tissue-level effects on wound healing have not been formally investigated.

Therefore, in the present study, the investigators examined the chronic healing response of wounds after hemostatic treatment with ES and SCBS in adult Yorkshire swine. This species was chosen because swine are a well-established model for most human surgical procedures and their use as a model for wound healing is well-documented.^2,7,24 Porcine organs, muscle, and bone are similar to those of humans in physiology and size, and the swine itself is an appropriate mass to model the adult human. Porcine skin possesses similar architecture to human skin, and the healing process occurs through physiologically similar phases (inflammation, proliferation, reepithelialization, and remodeling) in pigs and humans.²⁴

The results reported herein demonstrate that wounds treated with SCBS and ES exhibited a similar progression of healing during the 8-week study period. Both devices controlled bleeding as evidenced by lack of hemorrhage. However, wounds in muscle tissue treated with SCBS exhibited a greater depth of thermal effect compared with tissue treated with ES over the first 3 weeks, despite a markedly shorter treatment time required to achieve hemostasis.

The SCBS group exhibited a somewhat slower progression of tissue maturation over the course of recovery compared with the ES, as shown by higher (less favorable) scores for collagen deposition, fibrous tissue maturity, extracellular matrix, and stage of healing. However, at the end of the 8-week recovery period, there were no significant differences in healing parameters between the 2 groups.

Fewer inflammatory markers were observed with SCBS at early time points, but a consistent pattern was not observed. Myofiber regeneration was noticeably increased for the SCBS group over the ES group at weeks 2, 3, and 8.  This may indicate a better regenerative response to replacing damaged muscle tissue in the SCBS group. Yet, it cannot be established if this regenerative response was due to the greater depth of hemostatic effect of SCBS or if it confers some therapeutic advantage. 

These results may provide some insight at the cellular level into the healing activities underlying the positive results observed in clinical studies. However, it is not possible to determine the degree to which these results translate to patient clinical outcomes.

This study had several limitations. First, there was no prior research regarding chronic wound healing with the SCBS by which the authors could properly power this experiment for the endpoints of interest. They attempted to mitigate this limitation with a reasonably high number of animals and by increasing the number of test areas per animal; however, they were limited by the available dorsal anatomical space and overall impact on morbidity of the procedures.   

Second, the histopathological measurements were made by multiple veterinary pathologists. Although these individuals were trained to a standard methodology, blinded to the treatment modality, and passed repeatability and reproducibility measurement testing (ie, Gage repeatability and reproducibility), there is inherent variability to their observations. A single pathologist performing these measurements may have minimized variability; however, it would have materially delayed the conclusion of the study.  

Lastly, since wound healing is a complex, multistage process with overlapping phases, the measurement time points may not perfectly coincide with most clinically-relevant periods. 

Although the clinical significance of these results requires further exploration, this study demonstrates that the use of SCBS to achieve hemostasis resulted in a deeper tissue effect in a shorter amount of treatment time, with improved myofiber regeneration and a comparable course of healing, compared with hemostasis treatment using traditional ES.

The authors thank Jeanne McAdara, PhD, for professional assistance with manuscript preparation.

Affiliations: Medtronic Transformative Solutions, Portsmouth, NH; Medtronic ENT, Jacksonville, FL; and Medtronic Physiological Research Laboratory, Minneapolis, MN 

Correspondence:
Joshua G. Vose, MD, MBA
Senior Medical Director
Medtronic Transformative Solutions
180 International Drive
Portsmouth, NH 03801
joshua.vose@medtronic.com

Disclosure: All authors are employees of Medtronic, Inc (Minneapolis, MN).