Establishment of an Uncomplicated Radiation-delayed Wound Healing Model Using Irradiation in Pigs

Introduction. Radiation-delayed wounds require diverse therapeutic strategies to achieve effective healing. However, the development of novel therapies with a radiation-delayed wound healing model is hindered by the lack of standardized animal models. Objective. In this study, the authors propose and verify a procedure to establish a radiation-delayed wound healing model in pigs. Materials and Methods. Two female pigs received a single 18-Gy dose of a 6-MeV electron beam per 18 cm x 8 cm area. Three areas were treated on the paraspinal dorsal skin surface of each pig, with 2 on the left side of the spine and 1 on the right. Wounds were periodically created on the 2 pigs at 1 of the following time points: (1) 2 weeks post radiation (PR2 group; n = 4), (2) 4 weeks post radiation (PR4 group; n = 4), and (3) 6 weeks post radiation (PR6 group; n = 4). A partial-thickness wound was created by excising the skin, superficial fat layer, and superficial fascia while preserving the deep fat and deep fascia. Wound contraction was evaluated, and histological analysis was performed at 2 and 4 weeks after wounding. Results. The control wounds displayed complete reepithelialization at week 4. However, the PR6 group showed delayed wound healing for the entire experimental period. Furthermore, compared with the control group, the PR6 group demonstrated excessive acute and chronic inflammation and exhibited incomplete reepithelialization at week 4. Conclusions. These findings suggest skin wounding 6 weeks after irradiation is most suitable for the induction of a delayed wound healing model. Using this protocol, the authors safely generated a delayed wound healing model without acute complications from irradiation.

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Delayed wound healing requires diverse therapeutic strategies to achieve effective healing. However, the development of novel therapies using delayed wound healing models is hindered due to the lack of standardized animal models. Many impaired-healing models have been developed to observe the potential effects of experimental treatments.^1-4 Because of the similarities in the anatomy and physiology of wound healing between swine and humans,⁵ and because both have similar responses to irradiation time and dosage,⁶ swine typically are considered convenient animals for delayed wound healing models. 

In previous experiments, the authors developed a delayed wound healing model using mechanical compression and irradiation in pigs.^4,7 Initially, they attempted to create an ischemic wound using mechanical compression.⁴ However, this approach was limited by the prolonged placement of silicone blocks, which mechanically prevented wound contraction. Irradiation also was used to generate a delayed wound model in previous work.⁷ The authors had concluded from that investigation⁷ that irradiation alone was sufficient to create a delayed wound healing model.Irradiation affects normal skin and can cause skin necrosis depending on the dosage.^8-10 Furthermore, the irradiation effect varied over time.⁸ 

Therefore, the authors now attempted to develop a reliable radiation-delayed wound healing model without compromising normal skin, which could potentially bias the experiment. In addition, they sought to determine which time point for wounding is most appropriate to induce delayed wound healing after irradiation. 

Animal irradiation
Two female micropigs (Micropig; Medi Kinetics Co Inc, Gyeonggi-do, Republic of Korea) > 7 months of age, free from skin diseases, and weighing 30 kg to 32 kg were used. At 6 months of age, micropigs complete the development of secondary sexual characteristics and are sufficiently mature. With restricted feed, the pigs did not grow during the experimental period. These conditions permit convenient examination of the wound-contraction process. 

One week prior to the experiment, the pigs were moved from the breeding farm to the laboratory to allow acclimation. Each pig was housed in a separate cage and was given 400 g of standardized gamma-irradiated feed and 3 L of water daily. The laboratory was maintained at 21°C to 23°C with a relative humidity of 53% to 59%. The Keimyung University School of Medicine Institutional Animal Care and Use Committee (Daegu, South Korea) approved all experimental procedures.

On the day of irradiation, pigs were anesthetized with an intramuscular and intravenous injection of tiletamine-zolazepam (Zoletil; Virbac Laboratories, Carros, France) and xylazine hydrochloride (Rompun; Bayer, Lerverkusen, Germany). Before irradiation, a computed tomography (CT) scan was performed to assess skin thickness and to ensure an adequate irradiation depth. Each pig received a single 18-Gy dose with a 6-MeV electron beam per 18 cm x 8 cm area using a linear accelerator (Clinac iX System; Varian Medical Systems, Inc, Palo Alto, CA). Three areas on the paraspinal dorsal skin surface of each pig were irradiated: 2 on the left side of the spine and 1 on the right. Therefore, the irradiated zone was designed so that all of its margins bordered normal skin (Figure 1A). 

The radiation level was calculated to ensure that > 90% of the prescribed dose would be limited to a maximum depth of 2 cm. The skin in the caudal area was thinner than in the cephalic area (Figure 1B). To compensate for the thinness of the caudal skin, bolus material was used in the caudal area. The borders of the fields were delineated to obtain precise treatment areas. 

Following irradiation, the animals were transported to the animal lab and housed under standard conditions.

Wound creation
Wounds were periodically created in 2 pigs at 3 time points: 2 weeks after irradiation (PR2 group; n = 4), 4 weeks after irradiation (PR4 group; n = 4), and 6 weeks after irradiation (PR6 group; n = 4). The control wounds (control group; n = 6) were created on opposite sides of the spine in the non-irradiated zone (Figure 2). Each pig was anesthetized as previously described and moved to the operation room. After the hair was shaved, a square 4-cm² excisional wounding site in the irradiated zone was marked with an oil ink pen; the site was 2 cm from the boundary. Each wound was positioned 6 cm from the other. At each wound-creation time point, 2 irradiated-zone wounds and 1 control wound were made on each pig. After marking, the skin was sterilized with betadine, and wounds were accurately made with a No. 10 blade. Wounds were created above the superficial fascia and included the skin and the superficial fat layer. Each wound was separated by a preserved deep-fat layer. 

Wound healing rate
Raw surface areas were directly assessed at 1-week intervals for 4 weeks via digital planimetry (VISITRAK Digital; Smith & Nephew, Hull, UK) according to the manufacturer’s instructions. Briefly, a 2-layered plastic sheet was placed atop each wound, and a marker pen was used to accurately trace the contracted and epithelialized wound edges. The authors did not differentiate between wound contraction and epithelialization. The sterilized adhesive layer of the plastic sheet in contact with the wound then was separated from the upper transparent layer, which was placed on the planimetry device. To measure the wound surface area, the wound outline was redrawn along the marked line using a special stylus. 

Histological analysis
Biopsy of irradiated and normal wounds was performed 2 and 4 weeks after wounding. Samples were fixed using 10% neutral buffered formalin, embedded in paraffin and cut into 4-µm sections. Tissue sections were stained with hematoxylin and eosin (H&E) for general histological analysis.

Histological analysis was performed by a pathologist who was blinded to the experimental protocol. The levels of acute inflammation, chronic inflammation, and epithelialization were analyzed. The number of neutrophils and eosinophils were used as a marker for the severity of acute inflammation, and the number of lymphocytes, plasma cells, and giant cells were used as a marker for the severity of chronic inflammation.

Epithelialization was classified into 1 of 3 degrees: none; low, denoting epithelialization in less than half the wound; and high, denoting epithelialization in more than half the wound.

Statistical analysis
All data are expressed as the experimental mean ± standard error of the mean. The results were analyzed using the Kruskal-Wallis test with Dunn’s post hoc test using GraphPad Prism 5 (GraphPad Software Inc, San Diego, CA). Statistical significance was set at P < .05.

Gross findings
In the control wounds, the granulation tissue was fresh and grew quickly on the wound bed. The control wounds were filled with beefy-red-colored granulation tissue that covered the entire defect area 2 weeks post wounding. Reepithelialization was 82.9% 3 weeks after wounding. The wounds in the PR2 group contained unhealthy granulation tissue 2 weeks following wounding. However, reepithelialization 4 weeks after wounding in the PR2 group was 71.9%. In the PR4 group, the wounds demonstrated abundant discharge and scanty granulation tissue 1 week following wounding. After 2 weeks, the PR4 wounds were covered with dry crusts; they still were not completely filled with granulation tissue 4 weeks after wounding, demonstrating 47.8% wound contraction compared with the initial wound. Reepithelialization also was minimal throughout the experimental period. In the PR6 group, the wounds showed discharge and hard crusts like those of the PR4 group. Granulation tissue formation also was minimal until 2 weeks after wounding, demonstrating 40.6% wound contraction compared with the initial wound 4 weeks after wounding. Reepithelialization was not grossly detected, even at 4 weeks following wounding (Figure 3). 

Early in the 4-week observation period after wound creation, the wound healing rate was accelerated during weeks 1 and 2 in the control and PR2 groups. However, the PR4 and PR6 groups showed delayed wound healing throughout the experimental period. The wound-contraction rate in the PR4 group was significantly delayed, relative to that of the control group at 2 and 4 weeks (P < .05). The wound-contraction rate of the PR6 group also was significantly delayed, relative to that of the control group throughout the experimental period (1, 2, and 4 weeks, P < .01; 3 weeks, P < .001). However, no significant difference was found between the PR2 and control groups (Figure 4).  

Histological analysis of wounds
Reepithelialization was distinguishable according to the number of weeks after post-irradiation wound creation. The control group demonstrated a high degree of reepithelialization at 2 weeks and complete reepithelialization at 4 weeks. Although the PR2 group displayed a low degree of reepithelialization at 2 weeks, reepithelialization was more limited in the PR4 and PR6 groups at 2 weeks. The PR2 group showed a high degree of reepithelialization at 4 weeks. The PR4 and PR6 groups exhibited a low degree of reepithelialization at 4 weeks (Figure 5).

The degree of inflammation was determined by the number of infiltrating inflammatory cells. The control group showed mild-to-moderate acute inflammation and no chronic inflammation at 2 weeks. At 4 weeks, acute inflammation subsided, with only mild chronic inflammation in the control group. The PR2 group demonstrated marked acute inflammation and mild chronic inflammation at 2 weeks. At 4 weeks, acute inflammation subsided, with only mild chronic inflammation. The PR4 group displayed moderate acute inflammation and mild chronic inflammation at 2 weeks. At 4 weeks, acute inflammation was mild with moderate chronic inflammation. The PR6 group showed marked acute inflammation and mild chronic inflammation at 2 weeks. At 4 weeks, acute inflammation was mild with marked chronic inflammation (Figure 6).

In this study, the authors created a delayed wound healing model using irradiation without compromising normal tissue. These findings indicated that ionizing radiation with a single 18-Gy dose had a negative effect on wound healing and allowed delayed wound healing. This method was suitable for creating a skin defect 6 weeks after irradiation, thereby generating a radiation-delayed wound healing model, as indicated by the results of chronic wound inflammation and the wound healing rate. 

A chronic or nonhealing wound is defined as a wound that does not heal in a timely fashion or fails to progress through a normal sequence of repair in 4 to 8 weeks.^11,12 Although a popular chronic wound model (diabetic mouse) exists, this small animal demonstrates different wound healing characteristics, due to the panniculus carnosus, in comparison to swine, which shows similar wound healing properties to humans.^13-15 The authors previously investigated the development of a delayed wound healing model using mechanical compression and irradiation in pigs.⁷ In that study, they found irradiation alone was suitable to induce a delayed wound healing model.⁷ However, 2 cases of skin necrosis developed in that experiment,⁷ even without wounding (unreported data). In light of that, they adjusted the irradiation field and dose in an attempt to create a reproducible radiation-delayed wound healing model that would not compromise normal tissue. 

The irradiation effects were divided into 2 different categories, including acute effects that act on rapidly proliferating tissues and late effects that act on slowly proliferating tissue.¹⁶ Endothelial cells, which have a slow turnover rate, are damaged by late irradiation effects. Microvascular damage and occlusion developed due to endothelial cell loss, aggregate formation, and enlargement at 6 months after a single dose irradiation of 17.5 Gy to 25 Gy.¹⁶ Similarly, the microvascular count was decreased gradually and reached a nadir at 7 weeks in skin exposed to a single dose of 16 Gy or 18 Gy.⁸ The microvascular damage induced ischemia and resulted in impairment of wound healing. Therefore, the authors attempted to determine the time point after irradiation and the radiation doses that are sufficient to produce delayed wound healing. 

Skin irradiated with doses of 15 Gy to 30 Gy continued to recover from irradiation-induced injury, while skin irradiated with doses of 50 Gy to 75 Gy was unable to fully recover, as the initial damage was too severe.¹⁷ In their previous experiment,⁷ a single 20-Gy dose was delivered to each side of the paraspinal dorsal skin surface of the pig to generate a delayed wound healing model.⁷ Archambeau et al⁹ reported that pigs and humans respond similarly to radiation that does not result in skin necrosis at doses of 21.3 Gy.⁹ Furthermore, Hadad et al⁸ reported the microvasculature continuously decreased after a single 20-Gy dose of radiation. 

Based on those reports, the authors adopted a 20-Gy single radiation dose to create a radiation-delayed wound healing model. However, they observed skin necrosis even without wounding when a 20-Gy single radiation dose was utilized. An initial increase in the density of microvessels possessing distinct lumens in skin exposed to 18 Gy of radiation was followed by a dramatic decline in microvascular density, whereas 20 Gy of radiation provoked a continuous decrease of microvessels.⁸ In lieu of this, the radiation dose was decreased to 18 Gy to reduce the early damage to the microvasculature induced by a 20-Gy dose. In addition, they devised an irradiation field and wound-creation protocol to reduce early skin necrosis in the absence of wounding. In this experiment, each pig received radiation in a field measuring 18 cm x 8 cm, with a protected, nonirradiated area in both paraspinal regions. This design ensured the entire boundary of the irradiated field was surrounded by nonirradiated tissue. As a result, a minimal vasculature that would prevent skin necrosis could be guaranteed in the surrounding tissue, and immediate complications, such as lethality due to excessive irradiation, could be prevented. 

Furthermore, this irradiation-field design enables the irradiated wound to closely match the control wound in the cephalic-to-caudal direction. Wound healing of the porcine dorsum was observed to be different in the cephalic-to-caudal direction.¹⁸ Wound-contraction rates were significantly higher for cephalic wounds. Therefore, wound matching between the control and irradiated wounds in the cephalic-to-caudal direction is important for wound healing experiments in pigs. In previous studies, full-thickness wounds were made for chronic-wound-model research.^3,8,19 Because the subcutaneous tissue was detached easily from the fascial plane in the case of full-thickness wounding, the wound exudate could spread through the fascial plane and spread infections. In this experiment, only superficial adipose tissue was excised, whereas the deep adipose tissue was preserved to avoid the spread of infection through the fascial plane. When making a skin wound on an irradiated animal that is vulnerable to infection and extensive skin necrosis, care must be taken that the wound is not so close to others or so deep that it leads to wound merging and the spread of infection. 

Disordered and persistent inflammation is another cause of chronic or nonhealing wounds.^20-23 In this study, the authors used different inflammatory cells as markers of acute and chronic inflammation. The control group demonstrated the weakest acute and chronic inflammation during the experimental period. Although the PR2 and PR4 groups demonstrated mild-to-moderate chronic inflammation during the experiment, the PR6 group showed marked chronic inflammation at 4 weeks. The control group displayed a high degree of reepithelialization at 2 weeks and complete reepithelialization at 4 weeks. Although the PR2 group showed a high degree of reepithelialization at 4 weeks, the corresponding PR4 and PR6 groups demonstrated a low degree of reepithelialization. In agreement with the results for inflammation and reepithelialization, wound healing in the PR4 and PR6 groups was obviously delayed at 4 weeks compared with that in the control group. In particular, the PR6 group showed consistently delayed wound healing relative to the control group throughout the experimental period. In this study, wound creation 6 weeks after irradiation was sufficient to induce radiation-delayed wound healing in the pig. 

One limitation of this study was that the 4-week observation period was not sufficient to identify complete wound healing in all experimental groups. However, this experimental period may be sufficient to determine delayed wound healing because the minimum criterion for observing delayed wound healing is a 4-week delay. Furthermore, significant differences were observed in the inflammatory degree and wound healing rate between the experimental and control groups during the experimental period. 

Another limitation is the square wound shape. Because the wound contraction force acts in a centripetal direction, symmetrical wound contraction did not occur for the 4 sides, which resulted in corner artifacts. Therefore, further experiments using circular-shaped wounds are needed to decrease this variability. 

These study results suggest the most suitable method of creating a skin wound for the induction of a radiation-delayed wound healing model is to wound the skin 6 weeks after irradiation. Using this protocol, a radiation-delayed wound healing model safely, without acute complications of irradiation, was established. Thus, this experiment presents another delayed wound healing model that can be used by researchers to investigate delayed wound healing.

Authors: Xiao Yang, MD¹; Woonhyeok Jeong, MD, PhD²; Daegu Son, MD, PhD²; Youngwook Ryoo, MD, PhD³; Jinhee Kim, MD, PhD⁴; Youngkee Oh, PhD⁴; Sunyoung Kwon, MD, PhD⁵; and Dalie Liu, MD, PhD¹

Affiliations: ¹Department of Plastic and Reconstructive Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou, China; ²Department of Plastic and Reconstructive Surgery, Institute for Medical Science, Dongsan Medical Center, Keimyung University, Daegu, Republic of Korea;  ³Department of Dermatology, Institute for Medical Science, Dongsan Medical Center, Keimyung University; ⁴Department of Radiation Oncology, Dongsan Medical Center, Keimyung University; and ⁵Department of Pathology, Dongsan Medical Center, Keimyung University

Correspondence: Daegu Son, MD, PhD, Department of Plastic and Reconstructive Surgery, Keimyung University School of Medicine; 194 Dongsan-dong, Daegu, Rep. of Korea, 41931; handson@dsmc.or.kr

Disclosure: This work was supported by a research-promoting grant from the Keimyung University Dongsan Medical Center in 2014. The authors disclose no financial or other conflicts of interest.