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Global Veterinaria 14 (5): 699-706, 2015
ISSN 1992-6197
© IDOSI Publications, 2015
DOI: 10.5829/idosi.gv.2015.14.05.94236
Corresponding Author: Alaa Moustafa, Department of Surgery, Anesthesiology and Radiology,
Faculty of Veterinary Medicine, Kafrelsheikh University, Kafrelsheikh, El Geish street, 33516, Egypt
Tel: 0472231609 E-mail: alaa.moustafa@bristol.ac.uk.
699
A Preliminary Study on the Effectiveness of Glycerin
Magnesia Dressing on Second Intension Healing of Wounds on the
Distal Aspect of the Forelimb of Horses
Alaa Moustafa and Mohamed Marzok
Department of Surgery, Anesthesiology and Radiology, Faculty of Veterinary Medicine,
Kafrelsheikh University, Kafrelsheikh, El Geish street, 33516, Egypt
Abstract: Second intention healing of full-thickness limb wounds in the horse is subject to numerous
complications including excessive granulation. Various topical preparations are purported to avoid these
complications, but beneficial effects of these medications seen in other species or in vitro have rarely been
reproduced in equine limb wounds. Here we investigated whether application of Glycerin Magnesia (GM)
dressing improves measures of wound healing compared with a non-adherent, semi-occlusive dressing applied
to standard experimentally-created wounds on the distal aspect of the equine limb. A 2.5 X 2.5 cm wound was
created on the dorsomedial aspect of the proximal metacarpus on each forelimb. A GM dressing was applied
to one assigned limb as a treatment and a non-adherent, semi-occlusive dressing was applied to the
contralateral limb as an internal control. Bandages were changed every 3 days for 39 days. Granulation tissue
was scored and wound area measured every 3 days. GM treatment accelerated wound healing in each horse
such that complete closure was achieved on average 6.2 ± 1.5 days sooner than in control wounds (p < 0.05).
The GM wound dressing did not increase granulation relative to control at any time point. Although addition
studies using the GM dressing are needed to characterize the cellular and temporal effects on wound healing
and evaluate this dressing in a clinical equine environment, this preliminary study suggests GM dressing is a
useful adjunct to accelerate equine wound healing.
Key words: Glycerin Magnesia Wound Horse Wound healing
INTRODUCTION Several studies have attempted to identify strategies
Distal limb wounds are commonly encountered in Whereas some treatments have been shown to improve
equine clinical practice because of the horse’s wound healing [5-13] others have not [14-19] and no
environment, its athletic usage and tendency toward flight single preferred method to treat distal limb wounds exists.
behaviour when startled. Distal limb wounds in horses Wound healing in the equine distal limb may be improved
heal more slowly than wounds on other parts of the body by dressings that speed wound contraction rates without
because of a comparatively decreased blood supply, an impairing motion, improve epithelialization, limit the
increase in bone prominence and lack of deep supportive formation of exuberant granulation tissue, be cost
tissue, greater mobility over joints and predisposition for effective and easy to use and not cause pain on
bacterial contamination because of proximity to the application or removal.
ground [1, 2]. Additionally, distal limb wounds in horses Glycerin is one of the most widely used ingredients
tend to have more skin retraction, exuberant granulation in medical prescriptions [20]. It may be used on every part
tissue formation, slower epithelialization and earlier of the epidermis and mucous membranes [21]. When
cessation of wound contraction, partially attributed to an diluted to a concentration below 50%, it acts as an
ineffective, but persistent inflammatory response [3, 4]. emollient and demulcent, finding important applications in
to improve management of distal limb wounds in horses.
Global Veterinaria, 14 (5): 699-706, 2015
700
ointments and lotions [18]. Antiseptic preparations for cobra bite envenomation shows that its external
the. most sensitive areas of the body including vaginal, application can promote wound healing [32].
nasal, analgesic, dermatological and burn ointments and In the current study we aimed to determine if
jelliesare commonly made of water soluble bases application of GM dressing alters wound healing in the
compounded with glycerin [20]. distal limb of horses. Since magnesium sulphate is
Glycerin scaffolds were introduced as another choice hygroscopic, it pulls out the fluid from the edematous
of wound dressing and their production process was tissue across the skin or the mucosa, which acts as a
simpler, more energy efficient and saves time and money semipermeable membrane and the mixture is exothermic
compared to the freeze-dried scaffolds [21]. The producing heat that cause vasodilatation which facilitates
glycerolized full-thickness skin harvested from body reabsorption of the fluid from edematous tissue and
contouring procedures is clinically effective in burn and thereby reducing edema, whereas glycerin has a soothing
wound management in human being [22]. In the presence action on the tissue [33].
of regional coordination, it can serve as an abundant Our hypothesis was that application of GM dressing
source for skin banking in where cadaveric skin use is not would improve wound healing variables (decrease wound
legalized [22]. Another study demonstrates that the use area, decrease granulation tissue formation, resulting in
of glycerin hydrogel-based wound dressing significantly faster healing times) when compared with a non-adherent,
decreases the number of peristomal infections such that absorbent dressing on surgically created wounds on the
the frequency of dressing changes can be safely distal aspect of the forelimb (medial aspect of the proximal
extended to 7 days during the first week, making it a less metacarpal region) of horses.
labor-intensive and more cost-effective option for wound
management after percutaneous endoscopic gastrostomy MATERIALS AND METHODS
[23]. A high glycerin econtent hydrogel mask dressing on
post-laser resurfacing wounds is a better and suitable The study protocol was reviewed and approved by
alternative to the open technique wound healing [24]. the Animal Care Committee, Faculty of Veterinary
A glycerin based dressing with commercial honey can be Medicine, Kafrelsheikh University, in accordance with
used in the management of a recalcitrant diabetic foot Egyptian ethical codes for studies on experimental
ulcer [25]. In addition, glycerin can be used as a animals.
preservative for corneoscleral rim grafts used in the
treatment of peripheral corneal disease [26]. Glycerin, Chemicals and Drugs
vaseline and liquid paraffin cream (Dexeryl) is commonly Magnesium Sulphate: Epsom salt was obtained from
used as topical hydrating agent for the treatment of skin El-Mansorah Pharmaceutical Chemicals Co. (El-Mansorah,
xerosis [27]. Egypt).
Magnesium sulphate (epsom salt) has been used for
many years in treatment of superficial equine wounds and Glycerin: Pure glycerine was obtained from El-Gomhouria
infections effectively by establishing drainage, soaking Pharmaceutical Chemicals Co (Ameria, Cairo, Egypt).
the foot in an Epsom salt solution, poulticing the foot
until drainage has ceased and protecting the foot until the Glycerin Magnesia (33%): The mixture of 33% solution
hoof capsule defect has healed [28]. Another study has of GM was prepared by dissolving 330 g of Magnesium
shown that the use of warm water/Epsom salt soaks for Sulphate in 100 ml distilled water with heat and
one week reduces the magnitude of infection and cause thoroughly mixing. After completely dissolving the
the swelling to subside in Asian elephant [29]. Magnesium sulphate, glycerin was added to the mixture
It is possible to combine the glycerin with magnesia. slowly with heat and continued stirring up to a final
Glycerin magnesia (Magnesium sulphate and glycerine, volume of one litre.
GM) enhances healing in human diabetic foot ulcer by
reducing the bacterial loads in the area [30]. A comparison Animals: Clinically healthy adult horses (n = 5), free of
between the effectiveness of fresh alovera and GM scars or blemishes on the forelimb metacarpal region were
application based on the level of severity of phlebitis in selected for this study. There were all non-castrated
children has shown that GM is significantly more effective stallions aged 13–18 years (mean, 14.8 years), weighing
in treatment of mild and moderate phlebitis when 360–520 kg. Animals were housed in indoor stalls and fed
compared to fresh aloevera [31].A retrospective study on on a balanced ration of mixed grain with hay and ad lib
the effect of GM on treatment of necrosis after Chinese water.
Global Veterinaria, 14 (5): 699-706, 2015
701
Study Protocol: Horses were sedated intravenously with epithelial margin surrounding the wound, which was
detomidine hydrochloride (0.01– 0.02 mg/kg) and converted to cm by the software program. Wound area
butorphanol tartrate (0.01 mg/kg) (Dechra was measured 3 times and the mean wound area was used
Pharmaceuticals, Shropshire, Uk). A 5 cm × 5 cm area was for statistical analysis. Wound measurements were
clipped of hair on the medial aspect of both forelimbs over performed by A.M. Complete wound healing was defined
both proximal metacarpal regions in each horse. The area as convergence of the epithelial margin without any
was cleaned once with povidone-iodine solution and evidence of granulation tissue.
100% ethyl alcohol. At the edge of the clipped region,
7 mg/kg 2% mepivacaine hydrochloride (Alexandria Statistical Analysis: A mixed model multivariate analysis
pharmaceutical company, Egypt) was infiltrated locally. in SPSS Statistics (IBMS, v.21) was used to compare the
A 2.5 cm × 2.5 cm template made by using a metal square percentage difference in wound surface area between
was centred in the clipped regions and a full thickness GM-treated and paired control wounds relative to 3 day
wound was created by incising the skin within the metal (set at 100% for each wound). The model evaluated the
square template using a #22 scalpel blade. Subcutaneous main effects of treatment (GM or control), time (6 days to
tissue was sharply removed down. A gauze was applied 39) and treatment by time interaction. A post-hoc
with pressure to the wound until haemostasis was Bonferroni correction was applied to compare treatments
achieved. Wounds were left open without treatment nor at each time point. Horse ID was included as a random
bandaging for 3 days. From day 3, bandages were applied effect, accounting for pairs of wounds being from the
cleanly with the investigators wearing non-sterile same horse.
examination gloves. A 5X5 cm width of non-adherent A Wilcoxon signed rank test was used to compare
semi-occlusive dressing (El Mahalla Co., El Mahalla, granulation scores across all time points. Paired t-tests
Egypt) was immersed in GM. The dressing was centred were used to compare the time taken to complete wound
over the wound and secured with soft, elastic roll gauze closure and the time to maximum granulation between
(El Mahalla Co., El Mahalla, Egypt). A standard bandage GM-treated and control wounds.
of absorbent cotton (El Mahalla Co., El Mahalla, Egypt) P < 0.05 was considered statistically significant. Data
was then applied to the distal limb (REF). For the control is presented as the mean ± standard deviation (St Dev)
limb, the same non-adherent semi-occlusive dressing was unless otherwise stated.
applied to the wound, secured with soft, elastic roll gauze
and covered with the standard bandage. Horses were fed RESULTS
phenylbutazone (3gm) (Dechra Dechra Pharmaceuticals,
Shropshire, Uk) on the day of wound creation and 2 GM Accelerates Equine Wound Healing: The surface
additional days. No peri- or postoperative antibiotics were areas of each control and paired GM-treated wound from
administered. Bandages on both forelimbs were changed each horse were normalised to baseline (day 3, set at
every 3 days until the wounds were healed. 100%). As a proportion of their original size, GM-treated
Wound Healing Assessment: Wound measures were time point between 15 day and 39 day (Figure 1a, b).
collected throughout the 39-day study. Time of wound The greatest difference between GM and control wounds
healing was noted for those wounds not healed by 39 was observed on day 24 (GM: 75.9% ± 2.8% reduction in
days. At every bandage change, assessment of wound size, Control: 55.5 ± 3.6% reduction in wound size,
granulation tissue was graded using a previously- p < 0.001).
reported scale [13] (1 = below skin edge; 2 = level with The GM-treated wound reached complete closure
skin edge; 3 = above skin edge but not overlapping; before the control wound in each horse (Figure 1c).
4 = overlapping skin edge). Assessment of granulation GM-treated wounds achieved complete closure on
tissue was performed by 2 surgeons (A.M., M.M.) average 6.2 ± 1.5 days sooner than control wounds
throughout the study. Every 3 days, as the bandages (Figure 1c).
were changed, the wounds were digitally photographed
directly in line with the wound. Wound area was GM Does Not Result in Excessive Granulation During
measured using imaging software (Image J 1.46s, National Equine Wound Healing: Granulation of each wound was
Institutes of Health, Bethesda, Maryland, USA), using the scored every three days. All wounds received a maximum
wound template to calibrate the program and correct for granulation score of 3 at least once during the study, but
magnification, by outlining the granulation tissue- none of the experimentally-created wounds investigated
wounds were significantly smaller than control at each
Global Veterinaria, 14 (5): 699-706, 2015
702
Fig. 1: Glycerin magnesia accelerates equine wound healing. A) Wound area was measured every three days and
normalised as a percentage of wound area at the start of the study (day 3, set at 100%). P values relate to mixed
model comparison of absolute wound area on each day with Bonfrerroni post-hoc correction. Points represent
mean percentage of initial wound area ± St Dev. Trend lines represent linear (control) and second order
polynomial (GM) lines of best fit. B) Representative images of control and GM-treated wounds on days 0 and
24. C) The time to complete wound closure of experimentally-created wound was recorded for each horse. Time
to closure was compared between control and GM-treated by paired t-test. * p < 0.05, ** p < 0.01, *** p < 0.001
control versus GM-treated
Fig. 2: Glycerin magnesia reduces overall mean granulation score and delays peak granulation. A) Granulation of each
control and paired glycerin magnesia treated wound was scored every three days. Points represent mean ± St Dev
granulation scores. Trend lines represent two-period moving average lines of best fit. B) The time point at which
each wound reached the maximum granulation score was recorded and compared between control and GM-treated
wounds. All wounds reached a peak granulation score of 3 and all control wounds first received a granulation
score of 3 on day 15. *** p < 0.001
Global Veterinaria, 14 (5): 699-706, 2015
703
in this study showed excessive granulation overlapping environment, which is proposed to permit autolytic
with the wound margins (score 4) at any time point. debridement and promote epithelialization [34]. We did
Control wounds produced increasing quantities of not evaluate the population of cells at the surface of the
granulation tissue up to a maximum at day 15, which was wounds microscopically at any point during the study to
maintained for approximately nine days before partly characterize the cell population and correlate the cellular
decreasing towards baseline (Figure 2a). In contrast, effects of a moist environment with the observed changes
GM-treated wounds reached the same level of granulation in wound area or granulation score. In addition, different
13.8 ± 2.7 days later than control wounds (Figure 2b) and semi-occlusive dressings vary in their ability to absorb
fully returned to baseline by the end of the study exudate and, when saturated, can exhibit characteristics of
(Figure 2a). occlusive dressings, which have been shown to promote
DISCUSSION it is difficult to broadly compare semi-occlusive wound
The influence of GM dressing on second intention intrinsic and extrinsic factors that may impact their
healing of surgically created wounds on the dorsomedial effectiveness.
aspect of the proximal metacarpus in adult horses was Furthermore, GM dressings were associated with
evaluated. The GM dressing resulted in significantly slower increases in granulation tissue scores than control
decreased wound areas and reduced granulation tissue wounds, but the granulation tissue was more often scored
scores compared with the control dressing. In addition to as being level with the skin edge, which should be optimal
the significant effects of GM on wound area and for wound contraction and epithelialization. Although the
granulation tissue scores, complete wound healing times cellular mechanisms were not characterized, it appears that
were significantly lower in GM-treated wounds that the main GM treatment effects we observed could be
healed faster than the control wounds. attributed to reduced wound size, followed by inhibition
Studies investigating the effects of semi-occlusive of exuberant granulation tissue. It is known that excessive
dressings on healing time, granulation tissue formation inflammation can impair wound healing. Glycerin in high
and effects on inflammatory cells have produced mixed concentrations has a slight but definite anti-microbial
results [7, 14-19, 34]. Howard et al. [7] reported an average action [24], which may reduce inflammation. It also
healing time of 71 days using a synthetic semi-occlusive appears to have anti-inflammatory effects by influencing
dressing on 6.25 cm wounds created similar to our study, the inflammatory response to injury, at least in part
2
which was longer than non-adherent control dressing, but because glycerin’s strong negative charge binds to
significantly shorter than a synthetic occlusive dressing. extracellular matrix molecules and modulates the
The conclusion from Howard et al.[7] study was that inflammatory response [24, 35], which could conceivably
synthetic semi- and fully- occlusive dressing resulted in impede exuberant granulation tissue formation. The
prolonged healing compared with the control dressing. dressing never sticks to the wound and does not dry out.
Our results differ from Howard et al. [7] in that granulation By maintaining a moist environment, the chances of
tissue production was not increased with the GM contamination decrease and initiate immediate pain relief
dressing compared with control wounds. However, the [24].
control dressing we used was different from the control Human full thickness allografts have used glycerin as
dressing used by Howard et al. [7] which limits direct a simple preservative, as it is cost-effective and possesses
comparisons between studies. antibacterial and antiviral properties as well as
It has been shown that Semi-occlusive dressings suppressing allograft immunogenicity. A euro skin bank
have been noted to be most effective during the postal survey of 37 European burn centers found that
inflammatory and proliferative phases of wound repair 90% of responding burn centers used glycerin preserved
[7, 13]. Our results confirm that the GM Semi-occlusive allografts regularly [22, 36]. Further studies are needed to
dressing is indicated during the early phases of wound verify and elucidate the antimicrobial effects of GM
healing, as the wound area and granulation scores were dressings in wounds of the equine distal limb.
significantly different between groups primarily within the External application of magnesium sulphate is usually
first 25 days. adopted to reduce local inflammation and swelling in
Semi-occlusive dressings are thought to be clinical practice. Once the magnesium sulphate is mixed
advantageous because they allow oxygen exchange at the into glycerin, the glycerin can effectively prevent its
surface of the wound while maintaining a moist evaporation and extend its duration of action [32].
exuberant granulation tissue formation [7, 34]. Therefore,
dressings between products or wounds, because there are
Global Veterinaria, 14 (5): 699-706, 2015
704
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wound and thus maintain a moist environment congenial Growth Factor In Full-Thickness Skin Wounds Of
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