The Effect of Polyhexanide, Octenidine Dihydrochloride, and Tea Tree Oil as Topical Antiseptic Agents on In Vivo Microcirculation of the Human Skin: A Noninvasive Quantitative Analysis

Background. Antiseptics are indispensable for wound management and should focus not only on the efficacy in reducing the bacterial burden but also on how much they interfere in wound healing. In this study, the authors analyzed the direct effect of topical antiseptic agents on the microcirculation of intact human skin. Methods. The perfusion dynamics were assessed before, and 10 minutes after, the volunteers’ fingers of the right hand (n = 20) were immersed in the following solutions — octenidine dihydrochloride, polyhexanide, tea tree oil, and saline solution. The authors used the Oxygen to See (LEA Medizintechnik GmbH, Giessen, Germany) diagnostic device for noninvasive determination of oxygen supply in microcirculation of blood perfused tissues, which combines a laser light to determine blood flow, as well as white light to determine hemoglobin oxygenation and the relative amount of hemoglobin. Results. Tea tree oil (+19.0%) (B. Braun Melsungen AG, Melsungen, Germany) and polyhexanide (+12.4%) (Lavanid, Serag Wiessner GmbH, Naila, Germany) caused a significant increase in blood flow compared to the negative control (-25.6%). Octenidine (Octenisept, Schülke & Mayr GmbH, Norderstedt, Germany) showed a nonsignificant trend towards an increase in blood flow (+7.2%). There were alterations in the values of hemoglobin oxygenation and the relative amount of hemoglobin, but these were not significant. Conclusion. Perfusion is an important factor for wound healing. Therefore, it might be advantageous if antiseptic agents would increase blood flow. Tea tree oil and polyhexanide have a positive effect on skin blood flow and can therefore be used especially in critically perfused wounds, provided the adverse reactions and the antimicrobial efficacy are comparable.

Antiseptics are indispensable when it comes to wound management. Over the years, several antiseptics have gained currency such as octenidine dihydrochloride and polyhexanide. As cationactive substances, these lead to the disruption of the inner cell membrane of the anionic bacillus.¹ Besides synthetic products, essential oils are often used in the topical treatment of wounds. The use of tea tree oil is becoming widespread in Europe and North America.² Terpinen-4-ol, the main water-soluble component of tea tree oil, has antibacterial activity against a wide range of bacteria including Staphylococcus aureus, Streptococci, coagulase-negative Staphylococci, and coliforms.^3,4

The focus of antiseptics should not only be on their efficacy in reducing the bacterial burden but also on how much they interfere in wound healing, which is a dynamic process involving complex cellular and extracellular mechanisms. One aspect of wound healing is the blood supply. Without sufficient blood flow, wound healing can be delayed or stopped. Furthermore, a disturbed skin perfusion might cause a progression of the lesion. To date, many studies^4-7 have sought to analyze the efficacy of topical antiseptics in terms of bacterial reduction. Little has been published about microcirculation alterations as a result of local antiseptics. In the existing literature,^1,8,9 perfusion changes caused by local antiseptics were mainly described in animal models. Not much is known about the perfusion alterations due to local antiseptic agents in human skin in vivo. In this study, the authors analyzed the direct effect of topical antiseptic agents on the microcirculation of intact human skin.

Healthy volunteers without comorbidities (eg, diabetes mellitus and arterial hypertension) were recruited from hospital staff (ie, physicians and medical students). Before participation, an informed consent that enabled all study participants to understand and agree to the potential consequences and benefits of their participation was obtained. Twenty volunteers were enrolled in the study: 9 males (mean age 27 years, range 24–38 years) and 11 females (mean age 26 years, range 23–33 years). All participants were exposed to the test solutions. Exclusion criteria included smoking, vascular diseases, diabetes mellitus, arterial hypertension, and perfusion-altering medication. In order to decrease interference from ambient light, all measurements were taken in a dimly lit room. The room was quiet and at a constant temperature of 21°C. The volunteers were asked to avoid talking or making extreme movements, as these could lead to errors in measurement. All volunteers were asked to lie supine on an operating room table with their right arm lying on a surgical arm table.

Skin perfusion of the fore, middle, ring, and little fingers of the right hand was assessed twice:  The first set of measurements of these fingers was carried out following a 5-minute rest period during which the volunteers laid down on the operating table. They remained lying down for the whole experiment; therefore, all measurements were taken in the supine position. The first set of measurements represented the baseline. Subsequently, the fingers were immersed in the respective test solutions and the second set of measurement was carried out after a 10-minute break. The negative control (saline solution) was set as the difference of perfusion of the first and second measurements when the fingers were immersed in the saline solution. The researchers obtained a randomization by switching the order of the test solutions, which ensured that each test solution interacted 5 times with each finger.

Four solutions were tested in this study. No division into groups was performed; all volunteers were exposed to all solutions:

1. Saline solution 0.9% (0.9% Sodium chloride irrigation, USP, 500 mL NaCl, Braun 0.9%; B. Braun Melsungen AG, Melsungen, Germany).
2. Octenidine dihydrochloride (Octenisept, Schülke & Mayer GmbH, Norderstedt, Germany; 0.1% octenidine hydrochloride, supplemented by 2% phenoxyethanol, and other ingredients including 3-amidopropyl cocoate dimethylammonium acetate, sodium D gluconate, glycerol 85%, sodium chloride, sodium hydroxide, and purified water).
3. Tea tree oil test solution which consisted of 5% tea tree oil (no specific brand) and 95% Sodium chloride irrigation (NaCl, Braun 0.9%; B. Braun Melsungen AG, Melsungen, Germany).
4. Polyhexanide (Lavanid 1, Serag-Wiessner GmbH & Co., Naila, Germany); polyhexanide 0.02% and ringer’s solution: isotonic electrolyte solution composed of sodium chloride, potassium chloride, calcium chloride, and purified water).

All test solutions were equilibrated to 23°C and filled in unsterile medicine cups. The fore, middle, ring, and little fingers of the right hand were then immersed in cups with the 4 respective solutions according to the aforementioned method. 

Microcirculation was quantitatively assessed using the diagnostic device (Oxygen to See, LEA Medizintechnik GmbH, Giessen, Germany) for noninvasive determination of oxygen supply in microcirculation of blood perfused tissues. This device transmits continuous wave laser light (30 mW, 830 nm) and white light (20 W, 500–800 nm) to tissue, where it is scattered and collected on the skin surface with fibers from the probe. The movement of erythrocytes causes a Doppler shift, which in turn is detected by the laser light and analyzed by the diagnostic device. This Doppler shift caused by the movements of erythrocytes is then computed and displayed as the blood flow velocity. The detected laser signal also correlates with the number of moving erythrocytes in the tissue. The diagnostic device can register and use this information along with blood flow velocity to calculate the blood flow. Furthermore, it includes the tissue spectrophotometry using a white light. This light is reflected by erythrocytes at certain wavelengths (ranging from 500–630 nm) depending on their oxygen saturation. The characteristic hemoglobin absorption spectra allows the determination of hemoglobin oxygenation and of the relative amount of hemoglobin. All parameters were measured at the same time and via 1 probe at 2 mm depth.

Statistical analysis
For statistical analysis, the Wilcoxon signed-rank test for paired data was used to test the difference in skin perfusion. Statistical significance was set at 5% (P < 0.05). The analysis was performed with SPSS Version 22.0 (IBM, Armonk, NY). 

The 20 volunteers included in this study matched the inclusion criteria and provided informed consent. There were no adverse reactions (ie, skin irritation, allergic contact dermatitis, redness, pruritus, or rash). 

The authors obtained a negative control by measuring the perfusion of 1 randomized finger before and after its immersion in the saline solution. This negative control showed a mean difference in blood flow of -25.6% ± 30.7% and a mean difference in the relative amount of hemoglobin of -8.7% ± 12.9%, as well as a mean difference in the hemoglobin oxygenation of -9.3% ± 14.1% (Figure 1). 

A comparison of the polyhexanide group with the control group (saline solution) showed a significant increase in the blood flow (+12.4% vs. -25.6%; P < 0.05), whereas the relative amount of hemoglobin (-2.2% vs. -8.7%; P > 0.05) and the hemoglobin oxygenation (-10.6% vs. -9.3%; P > 0.05) did not alter significantly (Figure 2). 

After immersion in octenidine dihydrochloride, the blood flow did not increase significantly compared to the control group (+7.2% vs. -25.6%; P > 0.05). The relative amount of hemoglobin (-13.4% vs. -8.7%; P > 0.05) and the hemoglobin oxygenation (-6.4% vs. -9.3%; P > 0.05) did not alter significantly compared to the control group.

A comparison of the tea tree oil group with the control group showed a significantly higher blood flow (+19.0% vs. -25.6%; P < 0.05), whereas the relative amount of hemoglobin (+1.1% vs. -8.7%; P > 0.05) and the hemoglobin oxygenation (+1.5% vs. -9.3%; P > 0.05) did not alter significantly (Table 1). 

While the efficacy of topical antiseptics against microorganisms and their tissue compatibility in terms of cell growth inhibition and cell toxicity have been dealt with extensively in the literature,^8,10 there is little published research concerning the interaction of antiseptic agents and microcirculation. In particular, possible perfusion alterations as a result of local antiseptic agents in human skin in vivo are not known. However, perfusion is one of the most important factors for wound healing. Therefore, perfusion alterations of injured tissue could impair wound healing. The goal of this study was to analyze the direct effect of topical antiseptic agents on intact human skin microcirculation.

The diagnostic device was used in this study to quantify blood flow and the relative amount of hemoglobin. Measurements mainly represent the capillary-venous compartment, because hemoglobin is largely located in the capillary and postcapillary system of the microvascular bed.¹² The diagnostic device used only measures nutritive, microvascular vessels, because light is completely absorbed if the vessel diameter is greater than 100 µm.^12,13 The device has been proven to be a reliable and valid tool in various conditions such as microvascular flap monitoring,¹⁴ flap microperfusion,^15,16 limb perfusion at different angles,¹⁷ and microcirculatory changes of cold contact injuries.¹⁸

Tea tree oil is an essential oil obtained by steam distillation of the leaves and terminal branches of Melaleuca alternifolia in the Australian states of New South Wales and Queensland.^3,19 The use of tea tree oil is becoming widespread in Europe and North America.² Currently, it is marketed as a natural topical antimicrobial and anti-inflammatory in preparations at concentrations between 0.5% and 100%.² Terpinen-4-ol, the main water-soluble component of tea tree oil, has antibacterial properties against a wide range of bacteria including S. aureus, Streptococci, coagulase-negative Staphylococci, and coliforms.^3,4 The available literature^3,6,20 clearly showed tea tree oil has antibacterial, antifungal, antiviral, and antiprotozoal properties. However, not all of these activities were quantitatively analyzed in vitro and in vivo, and the present data are promising but inadequate.¹⁸

Using the diagnostic device, the authors found a significantly higher blood flow in the tea tree oil group compared to the control group (saline group). This is consistent with the existing literature, which describes that terpinen-4-ol has no effect on sensory nerves but instead modulates vasodilation and plasma extravasation.^6,19,21 This vasodilation is attributed to a relaxation of vascular smooth muscle, without any effect on the activity of the sympathetic nervous system.^21,22 Furthermore, the tea tree oil group tends to increase levels of hemoglobin oxygenation and a relative amount of hemoglobin. Disadvantages of tea tree oil as a topical antiseptic include the described irritant and allergic reactions. In Carson et al,⁶ a mean irritancy score of 0.25 was found for tea tree oil, based on patch testing on 311 volunteers. In the present study, there were no adverse reactions such as skin irritation, allergic contact dermatitis, redness, pruritus, or rash. 

In the polyhexanide group, there was a significant increase in blood flow compared to the control group. This result is consistent with the literature. Following polyhexanide application on cremaster muscle, Goertz et al⁸ demonstrated an increase in perfusion by increasing arteriolar diameter and functional vessel density. Such an arteriolar diameter increase was also shown on the skin of mice using intravital fluorescent microscopy.¹ An advantage of polyhexanide is low tissue toxicity, even in high concentrations.¹⁰ Furthermore, Daeschlein et al²³ described that polyhexanide does not inhibit reepithelialization and reduces the wall of necrosis after skin graft.²³

In the octenidine dihydrochloride group, the blood flow was not significantly higher than in the negative group. Contrary to the literature, Goertz et al⁸ also found no significant influence on arteriolar diameter, whereas an increased functional vessel density was observed. 

Polyhexanide has been proven to be one of the most efficacious antimicrobial agents. Furthermore, the authors have shown a positive effect on the skin blood flow. Henceforth, in daily practice they use polyhexanide, especially in critically perfused wounds such as burns, if a prolonged contact time is feasible. If an immediate effect is required, the authors use octenidine dihydrochloride, because it showed better antimicrobial efficacy than polyhexanide.^8,24,25

Despite careful planning, this study is not without limitations. The application of the sensor is relatively simple and the step-by-step setup of the computer is straightforward. Nevertheless, the sensor must be placed carefully, since shearing motions and excessive pressure can result in false measurements. Furthermore, the authors confined the possibility of flaw or interpersonal variability by employing a well-trained and experienced investigator (Sabrina Krauss, MD, resident for plastic surgery), who carried out all measurements. Additionally, microcirculation changes are dependent on the immersion time and might lead to different results if the measurements were carried out after a longer or shorter period of time. This study describes only skin perfusion dynamics in healthy volunteers. The results could be different in such wounds as skin defects caused by acute trauma, thermal injuries, and chronic diseases.

Perfusion is a crucial process for wound healing. Therefore, it may be advantageous if antiseptic agents increased the flow of blood. In this study, tea tree oil and polyhexanide showed a positive effect on skin blood flow; therefore, it can be used specifically in critically perfused wounds provided, of course, the adverse reactions and the antimicrobial efficacy are comparable.

Affiliations: Department of Plastic, Reconstructive, Hand and Burn Surgery, BG-Trauma Center, Eberhard Karls University, Tübingen, Germany; Department of Plastic, Reconstructive and Hand Surgery, University Hospital, University of Bern, Inselspital, Switzerland; and Department of Plastic, Hand, Reconstructive and Aesthetic Surgery, Helios Klinikum Wuppertal, University Witten/Herdecke, Witten, Germany 

Correspondence:
Jens Rothenberger, MD
Department of Plastic, Reconstructive, Hand and Burn Surgery, 
BG-Trauma Center, Eberhard Karls University,
Tübingen, Germany 
jens.rothenberger@gmail.com 

Disclosure: The authors disclose no financial or other conflicts of interest.