Mechanical Evaluation of Silicone Gel on Wound Healing of Rat Skin

Introduction. While most studies have reported on the clinical benefits of silicone-based products based on clinical observations, only a few studies have investigated the mechanical properties of silicone gel-treated wounds to determine if the use of silicone gel improves the mechanical recovery of wounds. Material and Methods. The dorsum of each of 44 Sprague-Dawley rats was divided into 3 regions. Two, 1-cm long cuts were made in 2 of the 3 regions and secured with a full-thickness interrupted suture. After surgery, antibiotic dressings and silicone gel were applied to the treated wounds (6nD) 3 times per week. Only antibiotic dressings were applied to untreated wounds (6n). The unwounded region served as the control. The skins were harvested after 1, 2, 4, and 6 weeks for tensile testing and histology. Results. There was no difference between the groups in a gross examination. However, the untreated groups had more inflammatory cells infiltrate the injury site at week 1, which caused more vulnerability of the wound site to external stress. At all time points, the silicone gel tended to have a higher recovery index than the untreated group, but the differences did not reach levels of significance. Conclusions. Application of silicone gel on wounds decreases inflammatory reaction and improves recovery index.

  Improving healing outcomes has long been an important goal in wound care. In the past several years a number of physical and chemical treatments have been discussed, including the use of silicone-based products, which have been widely used in the treatment of scars.^1-8 Recently, a topical silicone gel (Dermatix, Meda Pharmaceuticals Ltd, Takeley Bishop’s Stortford, United Kingdom) was introduced. The self-drying silicone gel is made of polysiloxane and silicone dioxide. The action mechanism of silicone therapy is likely to involve occlusion and hydration of the epidermal layer of the skin.^9-11   Several studies have evaluated scar assessment scales, such as the Vancouver Scar Scale and Patient and Observer Scar Assessment Scale, and shown that silicone gel has better clinical outcomes for decreasing the height of scars; dissipating erythema; and reducing the symptoms of pain, itchiness, and irritation.^2,4,12-16 Additionally, most patients experienced almost no side effects with the silicone gel. Although silicone gel is known to help improve wound healing based on histological and clinical evaluations, the mechanical properties of wounds treated with silicone gel remains unclear. To the authors’ knowledge, there is no report that evaluates the mechanical property of silicone gel-treated wounds up to 6 weeks. Therefore, this study was performed to determine if the use of silicone gel would improve the mechanical recovery of wounds and to observe the healing process for up to 6 weeks.

  Animal experiments, mechanical tensile tests, and histology were used for this study. The study and animal care protocols were approved by the Institutional Animal Care and Use Committee of the Laboratory Animal Center at National Cheng Kung University, Taiwan.   The subjects of the experiments were 44 male Sprague-Dawley (SD) rats between 6 and 8 weeks old. The rats were first anesthetized with intravenous injections of chloral hydrate 400 mg/kg (Sigma-Aldrich, St. Louis, MO). After anesthetization, the dorsum of each rat was shaved with electric clippers and then sterilized with 70% alcohol solution. The shaved area was divided into three 3 × 5 cm regions (control, silicone gel-treated, and untreated). Two, 1-cm long, full-skin thickness, incisional wounds were made in 2 of the 3 regions (Figure 1). Each wound was secured with a full-thickness interrupted suture using monofilament 6/0 nylon (UNIK, Taipei, Taiwan). Immediately after surgery, antibiotic wound dressing (Spersin ointment, Sigma Pharmaceuticals Pty, Ltd, Clayton, Australia) and silicone-based gel (Dermatix, Meda Pharmaceuticals Ltd, Takeley Bishop’s Stortford, United Kingdom) were applied to the silicone gel-treated (6nD) wounds 3 times per week until the subjects were sacrificed. For the untreated (6n) wounds, a regular antibiotic wound dressing was applied at the same time. The unwounded region, the tensile strength of which is used to normalize the tensile strength of the wound for each rat, served as the control. The rats were caged individually to avoid damage to the wounds.   Seven days after surgery, the nylon sutures were removed. At healing times of 1, 2, 4, and 6 weeks, the rats were euthanized with carbon dioxide, and skin samples were photographed and harvested for histological observation and tensile strength testing (Table 1 and continued).   A 2 × 5 mm cross section of skin, cut across the incision site, was obtained from the remaining tissue for histological testing. The sectioned tissue underwent classic specimen processing, including fixation, dehydration, infiltration, and embedding. The paraffin-embedded tissue block was sectioned with a microtome, and the sections were stained with hematoxylin and eosin (Sigma-Aldrich, St. Louis, MO). Picrosirius red stain (Polysciences Inc, Warrington, PA) for collagen fiber evaluation was also performed according to the manufacturer’s instructions.   A mechanical data collection instrument (Tytron 250, MTS Systems Corp, Eden Prairie, MN) was used in this study with soft tissue clamps used to grip the specimens. The mechanical testing process has been well established in previous studies.^17,18 In order to standardize the shape and dimension of the specimens and have the same geometrical effect during mechanical testing, biopsy specimens were acquired from the 3 regions of each rat and were flattened with a preload force of 0.2N applied on the clamped site. The thickness of all specimens was measured with a digital caliper. After being clamped to the mechanical data collection instrument, each specimen was subjected to 10 mm/min displacement at room temperature until failure (ie, breakage). The data were recorded at a sampling rate of 5 Hz. The force-elongation curve for each specimen was recorded, and the maximum force was identified as the breaking strength of the specimen. The tensile strength was calculated by dividing the maximum breaking strength by the cross-section area of each specimen. The recovery index was introduced to normalize the variations in the mechanical properties of each rat skin at different growing conditions, as reported by Levenson et al.¹⁹ The recovery index percentage was defined as dividing the tensile strength of the wounded skin by the tensile strength of the unwounded skin for each rat.   The results are presented as means ± standard deviations. The differences in the recovery index for all time intervals were analyzed by Wilcoxon rank sum testing. The Wilcoxon signed-rank test was used to compare the effects of different treatment methods at the same healing time when heterogeneity of variances or non-normality of the data was found. All statistical tests were conducted using the Statistical Package for the Social Sciences (SPSS) 13.0 for Windows at the 2-tailed test significance level of 0.05.

  Two rats died in the postoperative period due to infection, and 2 rat specimens were damaged in the routine experimental procedures. The average weights, tensile strengths, and recovery index of control, untreated wound skin (6n group), and silicone gel-treated wound skin (6nD group) at different healing times are listed in Table 1. A total of 40 rat samples were included in this study. Generally, the weights of the rats increased as the rats grew. The tensile strengths of both groups increased slightly with healing time, but significant differences were not found; however, after normalizing the variation of the size and condition of the skin, the recovery index of the treated groups and untreated groups steadily increased with time (Figure 2). A comparison of tensile strength and recovery index between treated and untreated groups found no significant difference at each healing time.   In gross examination, the wound edges were clearly observed in the first and second weeks, and there were better cosmetic outcomes in the treated groups than in the untreated groups. After 4 weeks of healing time, there was almost no difference in appearance between the 2 groups (Figure 3). The histology of the untreated (6n) group and the treated (6nD) group at different healing times is presented in Figure 4. Newly formed tissue can be distinguished in the dermis in both groups at all times. In the first week, many inflammatory cells infiltrated the injury sites in both groups. More inflammatory cells infiltrated the injury in situ in untreated groups than in treated groups in the first week (ie, up to 37 inflammatory cells in untreated groups vs 2 inflammatory cells in treated groups per high power field, 400X)(Figure 4a and 4b). These inflammatory cells quickly decreased, and almost vanished after 2 weeks of healing. Fibroblast proliferation with deposition of collagen fibers became predominant (Figure 4c and 4d). In this period, the healing strength was not strong enough to prevent occasional dehiscence (Figure 4c). After 4 weeks, the scars began to contract, and there seemed to be no significant morphological change between these 2 groups (Figure 4e-h). Picrosirius red stains examined under polarized light microscope revealed only a few green thinner collagen fibers in the first week. An increase in orange and red color in the collagen fibers were noted after the second week. After 4 weeks, the composition and structure of collagen fibers in the sutured area was similar to the adjacent nonsutured area. These changes were not significantly different at each time course between the 2 groups.

  In gross examination and histological results, no difference was found between the treated and untreated groups at 6 weeks. However, more inflammatory cells infiltrated the injury site in untreated groups at week 1. Also, the wounds in untreated groups were more vulnerable to external stress at week 2. Mechanical results showed that the treated group tended to have an approximately 8% improvement in recovery index at 6 weeks, (37.5% for treated groups, compared to 29% for untreated groups), but the differences did not reach levels of significance. The finding that silicone gel does not affect the tensile strength of sutured wounds may imply that it has no effect on the intermolecular cross-linking of collagen. In previous studies, similar results were also found regarding early wound strength, which indicate that there is little difference between the 2 groups on days 3, 5, and 7.²⁰ However, as the long-term effect of treated wounds is unknown and the mechanisms of silicone gel are still not clear, even from previous studies, this finding merits further investigation. Therefore, the authors conducted a study to examine the long-term effect up to 6 weeks.   Previous studies have been based on clinical observations,²¹ but few studies have focused on mechanical evaluation, except for one which investigated the effect of silicone-gel sheeting,²⁰ which is different from the silicone gel used in this study. The researchers immediately applied silicone-gel sheeting in acute wounds of pigs and performed mechanical evaluation. The biomechanical results showed there were no differences between the silicone-gel sheeting group and the control group at days 3, 5, and 7. Histological results showed little difference in conformation or composition change of collagen fibers. However, this study was designed to determine if silicone gel could improve wound healing at the healing time of 1, 2, 4, and 6 weeks.   The suture methods and the weights of rats may have influenced the healing process. According to previous studies,^17,19 recovery index may be up to 70 - 80% of unwounded strength on a 4-0 nylon-sutured incision at week 6. However, the recovery index in this study was only up to 29% on a 6-0 nylon-sutured incision at week 6. The 6-0 nylon sutures seemed unable to provide enough mechanical support for wounds compared to 4-0 nylon sutures, thus resulting in delayed healing. In addition, the weights of rats varied at different time points. This study showed the weights of rats increased with healing time. And the tensile strength in all groups also increased slightly with healing time. However, there is poor correlation between rat weight and tensile strength (Spearman’s correlation coefficient is 0.29). This finding is also consistent with the previous results.^18,19   There are some limitations to this study. One limitation was the uncertain dosage and duration of treatment for the rats treated with silicone gel. The suggested application for humans is to apply the gel twice per day; however, silicone gel was only applied 3 times per week in the rat model of this study. The characteristic of quick healing of rats and the physiological differences between rats and humans make the ideal dosage of gel treatment for rats difficult to determine. The dosage effects in rat models warrant investigation. In the future, monitoring the amount of silicone gel remaining on the wound and preventing the rats from rubbing off dressings are important issues. Previous studies have shown that the differences in scar quality between silicone gel-treated and control groups reach levels of significance that become apparent after at least 3 months follow-up. For example, van der Wal et al² investigated the effectiveness of silicone gel in promoting the maturation of burn scars at 1, 3, 6, and 12 months and found silicone gel-treated scars were significantly less rough and less itchy at 3 months. Chan et al⁷ assessed the effectiveness of silicone gel applied to patients who underwent a median sternotomy at 2 weeks, 4 weeks, and 3 months, and found significant differences between the control and silicone groups at 3 months. However, the quick healing rate of rats compared to humans may justify the 6-week observation period in this study.

  Based on the finding of the present study, the authors conclude that application of silicone gel on wounds decreases inflammatory reaction and improves recovery index.

Cheng-San Yang, MD, PhD is from the Department of Leisure, Recreation, and Tourism Management, Tatung Institute of Commerce and Technology, Chia-Yi City, Taiwan; and the Department of Plastic Surgery, Chia-Yi Christian Hospital, Chia-Yi City, Taiwan. Cheng-Hsin Yeh, MS is from the Department of Plastic Surgery, Chia-Yi Christian Hospital, Chia-Yi City, Taiwan. Chun-Liang Tung, MD is from the Department of Pathology, Chia-Yi Christian Hospital, Chia-Yi City, Taiwan. Cheng-Hsiang Jiang, PhD; and Ming-Long Yeh, PhD are from the Institute of Biomedical Engineering, National Cheng Kung University, Tainan City, Taiwan

Address correspondence to: Ming-Long Yeh, PhD Assistant Professor Institute of Biomedical Engineering National Cheng Kung University No. 1 University Road Tainan City, 701 Taiwan mlyeh@mail.ncku.edu.tw Disclosure: Financial support for this research was provided by the Chia-Yi Christian Hospital, Chia-Yi City, Taiwan.