The Effect of Topical Combination Therapy in Wound Healing: An Experimental Study

Objective. While topical therapies are common in wound care, the objective of this study was to evaluate the effect of combination topical therapy. Material and Methods. The authors designed a device that could irrigate and apply topical oxygen and topical negative pressure on the wound.  A round full-thickness excision wound was made on the backs of 30 rabbits, and the animals were randomly divided into 2 equal groups. The rabbits in the treated group received cycles of irrigation, negative pressure, and topical oxygen therapy 3 times a day for 60 minutes. In the control group, the wounds were irrigated with normal saline. The wound area was determined by image analysis software on days 1, 3, 7, 14, and 21 after the initial wound creation. The Mann-Whitney U test was used to analyze the data with SPSS 16.0 software. Results. On day 3 after wound creation, the percentage of wound closure in the treated group and control group was 30.1% and 3%, respectively, which shows the significant difference in healing (P = 0.0001). This trend was preserved in the rest of the study. Histological studies showed obvious enhancement in wound healing in the intervention group. Conclusion. The combination of the topical methods might have synergistic effects on wound healing.

  Wounds are a major health care burden. In 2002, global wound care expenditures were $13 to $15 billion.¹ It is estimated that 3-4 individuals per 1,000 develop acute or chronic wounds annually.² In 1996, approximately 11 million wounds were evaluated and treated in emergency departments across the United States.³

  Skin-wound healing is an orchestrated biological phenomena consisting of 3 sequential phases of inflammation, proliferation, and maturation.⁴ The process usually takes 3 to 14 days to complete.¹Although most acute wounds heal without a major problem,⁵ a considerable number of patients experience wound healing abnormalities.⁶ There is a growing need for a more specific and effective therapeutic agent to improve the healing process, especially in compromised wounds.⁵

  Through recorded history, wounds have been treated with topical medications and methods. Goals of topical wound management are speeding repair, preventing infection, reducing pain, removing dead tissue, providing a moist environment, reducing edema, and increasing blood flow.^7,8 Occlusive dressings, which maintain wounds in the moist state, are an effective choice for topical wound management.⁹ Topical negative pressure (TNP) therapy is another effective method for wound management.^1,8,10-14

  Therapies such as surgical revascularization and hyperbaric oxygen therapy have demonstrated that an improved perfusion and oxygenation of the wound accelerates the healing process. Topical oxygen therapy is a controversial issue. Some studies showed that topical oxygen therapy might enhance wound healing,^15-17 although some experts believe this therapy might not be effective.^1,7Irrigation is also a critical component of wound management, and is commonly performed with sterile normal saline solution or tap water.^3,18-23 In emergency departments, continuous wound irrigation with 100-300 ml normal saline or tap water with 0.35-0.56 kgf/cm² pressure generated by using a syringe, is recommended.³

  Finding new methods to improve wound healing can have a great value in clinical settings. In this research, the authors studied the simultaneous effect of some common topical methods in wound healing. A topical combination therapy delivery device was designed that could irrigate the wound and apply topical oxygen and negative pressure therapy as well. The goal of this study was to compare wound healing in the control group and in rabbits treated with the topical combination therapy delivery device.

 Ethical issues. This research was approved by the ethical committee of Kashan University of Medical Sciences, and was registered as an Iranian Randomized Clinical Trial (number 20119296228N3). All the procedures were in accordance with the Iranian guidelines for laboratory animals and the principles of laboratory animal care. The research was conducted at the Kashan University of Medical Sciences, Kashan, Iran.

  Experimental animals. In this interventional study, 30 mature male New Zealand rabbits weighing 4000-4500 g were housed in individual cages with controlled light, temperature, and humidity. The rabbits were fed commercial rabbit feed and water. Fresh carrots, cabbage, and lettuce were added to the commercial food.

  Wound creation. The animals were anesthetized by intramuscular injection of ketamin (40 mg/kg) and diazepam (4 mg/kg). The back of each animal, in the lumbar region, was shaved and prepared for aseptic surgery. A circular piece of skin with a radius of 2 cm was surgically excised, creating full-thickness type wounds.

  Experimental intervention. After wound creation, the 30 animals were randomly divided into 2 equal groups: treated group (n = 15) and control group (n = 15). The topical combination therapy delivery device had a plastic disc with an adhesive tape edge that could cover the wound. The plastic disc was held in place by an elastic band fastened around the rabbit. The disc was connected to the device by tubes.

  The device irrigated the wound with warmed normal saline (34 °C) with 0.45 kgf/cm² pressure. The pressure and temperature were adjustable and the irrigation continued for 3 minutes. Suction was simultaneously applied to the wound to collect the normal saline. After irrigation, a negative pressure of 90 mm Hg on the first day, and 100 mm Hg for the rest of the study, was applied to the wounds for 1 minute. At the end of the cycle, 100% warm oxygen (34 ºC) was applied to the wound area for 1 minute. The cycle of irrigation, negative pressure, and topical oxygen therapy was repeated for 60 minutes. The device was used 3 times a day, at 10 am, 2 pm, and 6 pm, in the treated group. In the control group, wounds were washed with normal saline manually, the disc was placed on the wound but the device was not turned on. The wounds were left open between treatments.

  After day 14, all the wounds in the treated group were covered by a crust and had satisfactory healing, so the topical oxygen therapy and negative pressure were stopped.

  Each rabbit had its own disc and pipes for treatment. After every use, the shield and pipes were decontaminated by soaking in 10% povidone iodine solution for 20 minutes and washed with soap and water before finally being cleaned with 70% alcohol and left to dry.

  Wound assessment. The wound area was measured by placing a transparent film on the top of the wound and labeling it with a marker before photographing with a digital camera. The wound area was calculated using image analysis software, and photographs of the wounds were also recorded. The images were assessed on days 1, 2, 7, 14, and 21 after initial surgery, and wound closure was compared with the control group.

  For histology assessments, skin samples were taken from the wound area and the margin on days 7, 14, and 21 following injury. The samples were fixed with 10% formalin and processed to prepare paraffin blocks, and serial tissue sections were stained with hemotoxylin-eosin (HE) or Van Gieson. In each microscopic section, 2 regions of healthy skin area and 2 regions of wounded skin area were examined under the microscope.

  The Mann-Whitney U test was used to evaluate the difference in wound area and wound closure. All analyses were performed using SPSS 16.0 software.

  Four rabbits in the control group and 3 rabbits in the intervention group died before study completion; these wounds were used in the comparison as long as the rabbits were alive, then the wounds in the rest of the rabbits were entered into the analysis. All the wounds were created in a similar manner, but widened a bit on their own based on the size and the posture of the rabbits during wound creation, so the final wounds were greater in size. The rabbits were randomly allocated to the treated or control group after wound creation, and Mann-Whitney test showed the wound area in the first day was not significantly different in the groups (Table 1). The wounds in the treated group healed faster than those in the control group (Table 1, Figure 1 and Figure 2). After 3 days, there was a 30.1 ± 8.7 percent reduction in wound area in the treated group, while in the control group it was only 3 ± 13.4 percent (P = 0.0001). After 7 days, the wound closure in the treated group was 48.7 ± 11 percent, compared to 20.3 ± 18 percent in the control group (P = 0.0001). There was no wound infection in any of the rabbits.

  In the study of skin sections stained with HE after 7 days, there was an intact epithelium and hair follicles in the healthy area. In this area of the dermis region, thick collagen fibers were stained red and fibrocytes were dense and fusiform; in comparison, the wounded area showed no sign of epithelium, hair follicles, or thick collagen fibers. Instead, the wounded area contained an abundance of red blood cells, fibroblasts, and inflammatory cells (Figure 3). Van Gieson staining showed collagen fibers as red with a yellow background, and the Van Gieson-stained slides clearly showed the different pattern of collagen distribution in the control and treated samples (Figure 3). In intact areas, collagen fibers were thick with fibrocyte within collagen bundles, but in wounded areas or healing regions, collagen fibers were in the form of thin and distributed fibers with fibroblasts between them. This specific staining verified the results deduced with HE slides.

  After 7 days, like the control samples, the treated slides clearly showed 2 healthy and 2 wounded areas. In the treated group, in addition to a thicker dermis, there was a growing epithelium in the wounded area, while in the control slides, there was no sign of the epithelium in the wounded area at this time (Figure 4).

  After 14 days, the control slides showed signs of growing epithelium in the wounded area. However, there was still dried blood in most of the wounded areas. In comparison to the control group slides, the slides from the treated group showed almost a complete epithelium on the wounded area. The dermis in the wounded area was thicker in the treated samples (Figure 4).

  Microscopic examination of the control and treated slides after 21 days showed no significant differences in terms of thickness of the dermis or epithelium in the healing region. In all 3 stages, the Van Gieson stained slides made it easy to compare the distribution of collagen fibers and of the epithelium (Figure 3).

  Wounds are associated with potential morbidity, impaired quality of life, and significant economic costs. Any improvement that speeds up the wound healing process has great clinical benefits for patients and health systems.²⁴ The results of this study showed a significant improvement in wound healing while using combination therapy. The topical combination therapy delivery device designed by the authors for the purposes of the study might be effective in full-thickness wound healing.

  Since the authors were not able to find a study with a similar device using a combination of 3 different parameters, results were compared with devices using 1 of the parameters. In this study, there was a 30.1% ± 8.7% reduction in wound area in the treated group after 3 days. Lee et al²⁵compared the wound closure in different hydrogel compounds in full-thickness wounds in rats. Wound closure ranged from 20% to 30% on day 3 after surgery,²⁵ which was comparable to the result in the treated group in the current study. Throusdale et al²⁶ showed 40% wound closure in full-thickness wounds in rats 7 days after surgery,which was lower than the 47.8% wound closure in the treated group 7 days after surgery in the current study.

  There are a wide range of topical management options in wound care. Topical negative pressure has been used as a method of wound management for some years,¹² and assists wound closure by applying localized negative pressure to the wound, promoting wound contraction and angiogenesis, and removing excess fluid.²⁷ Topical negative pressure has been suggested to be of value in a variety of settings, ranging from management of acute wounds to ischemic and diabetic ulcers.^27-29 Current indications for TNP dressings include pressure ulcers, venous ulcers, diabetic ulcers, and open wounds.^1,30

  A recent review summarizes the effect of TPN in clinical trials and confirms its benefit.8 Most of the studies included are clinical trials. In one trial study, 342 subjects were randomly divided into 2 groups to compare the efficacy of TNP with advanced moist wound therapy (AMWT) to treat diabetic foot ulcers. Results showed that a greater proportion of foot ulcers achieved complete closure with TNP (73/169; 43.2%) than AMWT (48/166; 28.9%) within the 112-day active treatment phase (P = 0.007).³¹ One of the outcomes of TNP in the referenced clinical trials was a reduced wound size, which the authors observed in the current study results as well.

  Irrigation is a critical component of wound management.3 Studies have documented the efficacy of normal saline irrigation in decreasing wound infection rates.^18,22 Wounds should be cleansed with a sufficient volume of irrigant to reduce or eliminate particulate matter and bacterial loads from the wound.²² Wounds traditionally are irrigated using a syringe and needle with manual injection of fluid, a time- and labor-intensive method.¹⁸ The designed device in this study can efficiently irrigate wounds with preregulated pressure, temperature, volume, and time. Further studies are needed to determine the advantage in clinical settings.

  The goal of oxygen therapy for wound care is to transfer sufficient oxygen to interstitial tissues to maintain a concentration near the 40 mm Hg found in healthy and well-perfused tissues.¹⁵ It has been commonly thought that skin receives its oxygen supply from internal circulation; however, recent investigations have shown that a significant amount of oxygen may enter the skin from the external overlying surface. It has now been shown that the oxygen penetration through > 700 (µm) of live human dermis can be achieved with both gaseous and topical dissolved oxygen delivery devices.¹⁵ There is currently some debate about using topical oxygen therapy in wound care. Many believe that topical oxygen therapy, which involves inserting the wounded limb into an airtight bag filled with oxygen under slightly elevated pressure, is clinically ineffective.1 However, some studies show that topical oxygen could improve the healing of venous ulcers, reduce recurrence rates, and alleviate pain.¹⁷

  Today, the principles of topical wound therapy involve elimination of necrotic tissue, control of bacterial loads, management of wound exudates, maintenance of open proliferative wound edges, and provision of a moist and protected wound surface.³² A single topical approach might not be able to achieve all the goals in wound care. The combination of therapies might accelerate the healing process. Hard-to-heal and sloughy wounds might benefit more from topical combination therapy.

  Most research focuses on the use of one topical treatment in wound care. The authors believe that a combination of topical methods (ie, irrigation, negative pressure, and oxygen therapy) might have synergistic effects and improve wound healing, and the results of this study support this assumption. More studies, especially in human beings and in clinical settings, are needed.

Rasoul Mokhtari, RN is from Student Research Committee, Kashan University of Medical Sciences, Kashan, Iran. Sayyed Alireza Talei, MS; and Mehdi Nouredini, PhD are from Physiology Research Center, Kashan University of Medical Sciences, Kashan, Iran. Negin Masoudi Alavi, PhD; and Tahereh Mazoochi, PhD are from Trauma Nursing Research Center, Kashan University of Medical Sciences, Kashan, Iran. Aliakbar Taherian, PhD is from Anatomical Research Center, Kashan University of Medical Sciences, Kashan, Iran.

Address correspondence to:
Negin Masoudi Alavi, PhD
Kashan University of Medical Sciences
Trauma Nursing Research Center
IRAN/Kashan
Ghotb Ravandi Highway
Kashan University of Medical Sciences
masoudialavi_N@kaums.ac.ir
alavi.negin@yahoo.com

Disclosure: The authors acknowledge this study was supported by the Research Deputy of Kashan University of Medical Sciences through grant no. 9022.