This study evaluated the use of an aliamide-containing gel in the treatment of skin wounds by employing noninvasive wound volume measurements acquired from wound images of high resolution ultrasonography (HRU). The dorsal thoracolumbar areas of 10 Beagle dogs were clipped and 2 rows of 6 full-thickness 5-mm punch biopsy samples were taken from either side of the midline under general anaesthesia. Wounds were allowed to heal by secondary intention, while treatment gel or base was applied 3 times daily to randomly selected treatment and control groups. Concentric 8-mm punch biopsy samples of the healing wounds were taken on Days 1, 2, 4, 8, and 14 for histopathological assessment. Wounds were imaged with 20 MHz HRU daily over a period of 28 days. Wound volume was calculated using scanner software based on the disk summation method. The wound volume was calculated in longitudinal and transverse ultrasound images and the average of the 2 values was used as the final wound volume. Repeated measures analysis of variance (ANOVA) was used to assess whether wound volume differed significantly over time, and between control and treated animals. Mean wound volume decreased significantly during the 28-day experimental period, and there was a significant time-treatment interaction indicating that the treatment effect varied at different stages during the healing process. Overall, application of the aliamide-containing gel appears to improve wound healing over time.

Disclosure: Funding for this work was provided by the Royal Veterinary College Internal Grant Scheme and by Innovet Italia Srl.

       Treatment of skin wounds in pets is a very common activity for practicing veterinary surgeons. Existing therapies for skin wounds take the form of surgical reapposition or grafting to close large skin defects, and the use of barriers and antimicrobial agents that maintain normal wound healing biology rather than accelerating and improving wound healing.¹ With improved understanding of wound biology comes the possibility of studying agents that directly promote optimal healing by using established techniques in animal models.2

Figure 1

       Mast cells probably play a pivotal role throughout wound healing, from the initial clot formation to the subsequent development of granulation tissue, re-epithelization, angiogenesis, matrix deposition, and remodeling by means of a piecemeal degranulation of their mediators, such as vasoactive amines (histamine, serotonin), cytokines (interleukins, tumor necrosis factor), growth factors (stem cell factor, platelet-derived growth factor, fibroblast growth factor, transforming growth factor), proteolytic enzymes (chymase, tryptase), proteoglycans (heparin), neuropeptides (nerve growth factor [NGF]), chemotactic factors (eosinophil chemotactic factor, neutrophil chemotactic factor, interleukin 1) and many others.³ It has been suggested that deregulation of mast cell degranulation could lead to a delay of wound healing and/or keloid and hypertrophic scar formation.^4–6
       N-acylethanolamines are a class of naturally-occurring aliamides that accumulate in injured tissues,^7,8 where they are believed to limit the inflammatory cascade and locally protect cells and tissues.⁹ Synthetic analogues of N-acylethanolamines have been produced, which down-modulate mast cell degranulation¹⁰ through a mechanism named “autacoid local injury antagonism” (ALIA).¹¹ For this reason, they are also called aliamides. Aliamides behave as endogenous agonists for a peripheral cannabinoid receptor on mast cells¹² and are able to reduce edema formation and tissue inflammation¹³ by down-modulating mast cells.¹⁴
       Adelmidrol, the International Nonproprietary Name (INN) of the diamide derivative of azelaic acid (N,N’-bis-(2-hydroxyethyl)-nonandiamide), is one of these synthetic analogues. It has been included in a re-epithelization gel coded ADL 20, currently under development by Innovet Italia srl (Milan, Italy) as a topical aid to wound healing. As previously stated, studies have indicated that deregulation of mast cell degranulatory function can lead to a delay in wound healing.^4–6 Thus modulation of mast cell degranulation in wound tissue by means of autacoids has the potential to speed up wound healing and lead to a better cosmetic outcome.
       Non-interventional wound healing evaluation for the assessment of treatment effects is currently a challenge. Various methods for assessment of wound area and volume have been evaluated.¹⁵ Wound area can be evaluated by currently available methods including the tracing of the wound with planimetry and digital photography with image analysis, but they are all limited to surface and surrounding tissue estimation. Volume assessment can provide a more complete picture of wound healing either by using a substance to fill the wound or by laser beam technology. The problem with such volume measurement techniques mainly lies with the interference with the healing tissue and the fact that none of these methods can be applied when a crust has formed or when the epidermis has regenerated sufficiently to cover the healing wound.¹⁶
       Ultrasonography is a relatively new technique that has been shown to be useful in the evaluation of the wound and wound healing without interfering with the healing process.¹⁷ This has been made possible by the development of high frequency ultrasound equipment, typically above 20 MHz, that allows visualization of the living epidermis, dermis, hypodermis, and deep fascia at a microscopic level and the term ultrasound biomicroscopy has been introduced.¹⁸ However, until recently, this method was not considered suitable for measuring wound volume as it involved making assumptions about the shape of the wound and the use of linear measurements that may have resulted in significant errors in volume calculation.¹⁷
       Computer software that measures wound volume using ultrasound images and the disc summation method has been used extensively in echocardiography and is considered to be one of the most accurate methods of measuring wound volume.¹⁹ Such software has now been developed for the assessment of wound biomicroscopy images based on 2-dimensional ultrasonographic images of cutaneous wounds. The aim of this study was to examine the effect of a topical gel containing Adelmidrol on experimentally produced epidermal and dermal skin wounds by means of wound volume calculation using this software with data generated during a previous study.¹⁷

Materials and Methods

       Creation of the wounds, their treatment and ultrasonographic measurement have been described previously.¹⁷ Briefly, 2 rows (duplicate sites) of six 5-mm punch biopsies (12 total) were taken from the dorsal thoracic area of 10 healthy Beagles and allowed to heal by second intention. Two 8-mm punch biopsy samples were taken at the site of the healing wounds (1 treated and 1 control) on Days 1, 2, 4, 8, and 14 (for a total of 10 biopsies for each dog) while the last 2 wounds were left in order to determine total healing time. Gel containing 2% N,N’-bis-(2-hydroxyethyl)-nonandiamide (ADL20, Adelmidrol Gel, Innovet Italia srl, Milan, Italy) was applied locally to the treated wounds 3 times a day. The same gel without the active substance (N,N’-bis-(2-hydroxyethyl)-nonandiamide) was similarly applied locally to the control wounds. A noninvasive, high-resolution (20 MHz) ultrasound biomicroscopy portable scanner (Longport Digital Scanner [LDS1], Longport International Ltd, Silchester, Reading, UK) was used to examine the wounds longitudinally and transversely daily by applying the transducer to the wound area using the applied active or inactive gel as the sound transmitting medium. All wounds were imaged until the second biopsy but only data from Day 6 to 27 were suitable for analysis.¹⁷ The 2 wounds that were not biopsied for a second time were examined throughout the study. Wound morphometry was performed on both the longitudinal and transverse digital HRU images.
       Wound volume measurement. Measurement of wound volume was performed on the detailed cross-sectional images of the skin wounds obtained by ultrasound using a program incorporated into the scanner software (EPISCAN, Longport International Ltd, Silchester, Reading, UK). The software allowed the wound margin of the 2-dimensional image to be traced and the volume to be estimated by adding together the volumes of a stack of discs, 1 pixel thick, whose diameters are lines drawn horizontally across the shape of the wound (disk summation method, Figure 1). In this calculation the axis of revolution of the whole shape will not always be a straight line. The software assumes that the wound being measured has a circular cross section in the horizontal plane. The wound volume calculations were performed on the longitudinal and transverse images from each wound per day and the average of the 2 measurements was used as the final wound volume.
       Statistical analysis. Data were analyzed initially for the entire experimental period (Days 6–27), and then subdivided into a preliminary phase (Days 6–12), during which the treatment was expected to delay healing, and a secondary healing phase (Days 13–27) with each phase being analysed separately. Descriptive statistics were obtained for the dependent variable wound volume. Data were examined for normality using the Kolmogorov-Smirnoff test and for equality of variance using the Levene’s test. The dependent variable was square-root transformed thereby allowing parametric tests to be performed on the data. Repeated measures ANOVA were used to assess whether wound volume differed significantly over time and between control and treated animals. The most appropriate covariance model was selected using Akaike’s information criterion. Post-hoc tests were performed using Bonferroni’s adjustment. P < 0.05 was considered significant. Results are presented as mean (standard deviation [SD], 95% confidence interval) unless specified otherwise. Analyses were carried out using SAS 9.1 for Windows (SAS Institute Inc, Cary, NC) and SPSS 13 for Windows (SPSS Inc, Chicago, Ill).

Figure 2

Results

       Over the entire experimental period (Days 6–27) there was a significant decrease in mean wound volume over time (P < 0.001) and a significant time*treatment interaction (P < 0.001) suggesting that treatment modified the effect of time (Figure 2). For the preliminary phase (Days 6–12) there was a significant difference (P < 0.001) between the mean volume of control (117.89 mm³ [SD 49.41 mm³, 95% CI 108.08–127.69]) and treated wounds (150.28 mm³ [SD 48.25 mm³, 95% CI 139.33–161.23]), but no significant change in mean wound volume over time (P = 0.4), as opposed to Day 6 when overall wound volume was 121.46 mm³ (SD 49.54 mm³, 95% CI 107.94–134.98) and 115.54 mm³ (SD 39.89 mm³, 95% CI 95.04–136.06) by Day 12.
       For the secondary phase (Days 13–27) mean volume of control [84.76 mm³ (SD 45.88 mm³, 95% CI 75.93–93.60)] and treated wounds [59.51 mm³ (SD 43.01 mm³, 95% CI 49.18–69.84)] differed significantly (P = 0.05) and there was a significant decrease in mean wound volume over time (P < 0.001) from 115.54 mm³ (SD 39.89 mm³, 95% CI 95.04–136.06) on Day 12 to 46.42 mm³ (SD 29.94 mm³, 95% CI 30.46–62.37) on Day 27. Neither group of wounds was completely healed by Day 27; measurements from the final day of the experiment showed mean volume of control wounds to be 57.42 mm³ (SD 41.07 mm³, 95% CI 19.44–95.41) and for treated wounds 37.85 mm³ (SD 15.07 mm³, 95% CI 26.27–49.43).

Discussion

       Previous studies evaluating wound healing have been limited by the lack of techniques that would allow 3-dimensional, serial evaluation of healing without interference with the wound. The development of high frequency ultrasound scanning has eliminated this problem. Ultrasonography allows serial evaluation of the wound without interfering with the healing process or the need for biopsy. It allows identification of the wound and can monitor the reduction of wound size over time.¹⁷ The limitation of ultrasonography was the lack of techniques for accurate evaluation of the wound volume without making assumptions that could influence the outcome. As a result, researchers have preferred to use the area of the wound as an indicator of wound healing.
       The incorporation of volume-measuring software that uses the disc summation method for evaluation of wound volume, into the ultrasound equipment, has provided the potential for more accurate evaluation of wound volume with minimal assumptions. The disc summation method has been used most often in echocardiography for estimating wound volume and is considered one of the most accurate techniques available.¹⁹ Since this software calculates the volume of the traced wound as a stack of discs, each 1 pixel high with a perimeter calculated by the tracing of the wound circumference, it provides the ability to measure wound volume without making any significant assumptions about the shape. Although errors in wound volume estimation are to be expected, these should be minimal and the comparison of both treatment and control wounds in the same fashion should significantly limit any effect on the results. The fact that ultrasound allows visualization and tracing of the wound in several orientations allows the examiner to make multiple estimations of volume and average the results, thus reducing the level of error in the volume calculation that may exist due to the unpredictable shapes of wounds in different orientations.
       Existing treatments for skin wounds rely on surgery and on procedures that maintain normal wound healing biology rather than modifying the processes involved in wound repair.¹ Aliamides are synthetic analogues of endogenous N-acylethanolamines, able to down-regulate mast cell degranulation by a mechanism called “autacoid local injury antagonism.”^10,11 This mechanism has been proved to be effective in cats given the oral aliamide analogue Palmidrol by means of mast cell densitometry.¹⁴ A similar inhibition of mast cell degranulation has been shown to occur in canine wounds treated by the Adelmidrol gel used in the present study, by means of histologic and electron microscopic investigations.²⁰ Based on results from these and previous studies^4–6 the authors’ hypothesis was that modulation of the mast cell degranulation in wound tissue by the local application of the aliamide Adelmidrol can potentially speed up wound healing and provide a better outcome. There is now increasing evidence that mast cells play a pivotal role in wound healing, both in healthy and sick patients. They are considered to be major biosynthetic sites of substances triggering inflammation and wound healing.^21–24 As sources of multifunctional cytokines,²⁵ growth factors,²⁶ and neurokines, such as NGF,²⁷ activated mast cells can affect other cell types both in the first phase of inflammation and in later phases of proliferation and remodeling.^28,29 Recent research by Weller et al³⁰ confirms that mast cells are required for normal healing of skin wounds and demonstrates that cutaneous wound healing responses are critically controlled by activated mast cells. Their study leads to the conclusion that after skin wounding, mast cells promote increased extravasation, polymorphonuclear neutrophil influx, and normal wound closure.³⁰
       In the present study, the treated wounds initially had a larger volume than the controls, suggesting that in the first phase of treatment Adelmidrol did not decrease wound volume. However, during the period between Day 12 and Day 27 there was a statistically significant difference between the groups, with the treated group healing faster and resulting in smaller wounds at the end of the study. This suggests that Adelmidrol enhanced overall wound healing by slowing down the process in the first phase. Wound healing is a complex process deriving from the interplay of a large array of cells, vessels, nerves, and biologically active mediators. It occurs as a sequential cascade of overlapping processes that requires the coordinated completion of a variety of cellular activities including phagocytosis, chemotaxis, mitogenesis, and the synthesis of matrix components. These activities do not occur in a haphazard manner but rather in a carefully regulated and systematic cascade. The resulting integrated wound healing networks are sequential as well as tightly controlled. The regulation of these events is multifactorial. It may be speculated that Adelmidrol promotes wound healing by controlling the initial release of mast cell mediators and thus slowing the opening phases of wound healing. The hypothesis is consistent with previous data showing that when the repair process is delayed the outcome of the healed wound appears to be improved and decreased scarring is observed.³¹ In contrast, it has been recently shown that sympathetic denervation, which accelerates wound contraction and mast cell differentiation, impairs re-epithelization and disturbs inflammatory and mast cell accumulation.³²
       In conclusion, the availability of high resolution ultrasonographic equipment with volume calculation software provides the opportunity to perform serial evaluation of wound healing without interference with the healing process. The tested aliamide may have a positive effect in wound healing and this may lead to more controlled healing and possibly better cosmetic outcome. More studies are required to confirm this.

Acknowledgement