Technical Bulletin September 2004 TB14-04

EpiThera®, a topical spray that promotes healing of surgical incisions, mechanical wounds and abrasions, and burns, has proven to be a highly effective anti-bacterial and antifungal topical spray effective against common bacterial and yeast pathogens such as Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Bacillus cereus, Candida albicans and Streptococcus agalactiae (See Lucid Med Tec II, Ltd. Technical Bulletins TB10-04 and TB11-04, accessed at www.lucidmedtec.com). TB11-04, entitled "Wound Healing and EpiThera®": Emu Oil's Permissive Effects on the Anti-Bacterial Actions of Benzyl Alcohol, addresses one of the underlying modes of action of EpiThera®on healing tissues. TB10-04 reported on the first of a series of experiments based on experimental skin healing of the rat, carried out by veterinarians specializing in wound care at The College of Veterinary Medicine at the University of Missouri-Columbia.

Wound healing is an involved process with a well-defined time course requiring several sequential events: inflammatory phase, proliferative phase, and maturation/remodeling phase (Pope, 1993; Hosgood, 2003). The present Technical Bulletin continues the examination of the efficacy and mode of action of EpiThera®on wound healing in rats, and specifically investigates the effects EpiThera® on the maturation/remodeling phase and the rate of eschar (scab) disappearance following controlled delivery of a skin wound.

The methodology for this study involved the humane creation of superficial skin burns in rats, after which the time course and gross anatomical appearance of the healing of these wounds was recorded in either groups receiving treatment with EpiThera®or with modified formulations of this product without emu oil (Details of these experiments' methodology and findings regarding beneficial benzyl alcohol-emu oil interactions are also provided in Lucid Med Tec II, Ltd. TB11-04).
Eighty male rats weighing 260-300 grams were individually housed in an animal care facility approved by the Association for Assessment and Accreditation of Laboratory Animal Care. All procedures were, in addition, approved by the Institutional Animal Care and Use Committee of the University of Missouri-Columbia. Each rat was anesthetized (isoflurane delivered in O2) and the upper surface of the trunk was then shaved and prepared for aseptic surgery by first scrubbing with chlorhexidine solution (The Butler Co., Columbus, Ohio) and then applying 0.05% chlorhexidine solution to the bare skin. To clearly identify the original site of burn induction, a permanent 2cm X 2cm grid was placed on the skin using a tattoo marker. A CO2 Laser (10 watts, 0.2 exposure time) was then used to create 2 cm2 partial-thickness wounds after the technique of Yang et al, 2001. Partial thickness wounds disrupt the entire epidermis and varying amounts of dermis, and commonly result from burns (to second degree) or severe abrasions (Pavletic, 1999). To ensure the comfort of the animal in the immediate post-operative recovery period, analgesia was provided by subcutaneous buprenorphine (0.1 mg/kg).

Technical Bulletin TB11-04 described a subset of this study focused on inflammation responses. In the phase of the studies described in this bulletin, two sets of rats were randomly placed into one of four treatment groups described below:

Group A. - emu oil alone
Group B. - EpiThera® formulation
Group C - EpiThera® formulation without emu oil
Group D - no treatment received

Only rats receiving the same treatment were housed together in cages, to prevent any cross-contamination.
The experimental protocol consisted of twice-daily application of 0.5 mls of the appropriate group treatment compound (described above), for the duration of the 21 day study. Wounds were left unbandaged.
Daily visual and photographic assessments were made of the burn wounds and their healing, noting in particular the presence and extent of eschar (scab) retention.
The effects of treatment group and time after surgery were assessed using a two way ANOVA, with a significance level of P<0.05.
This study was conducted as a blind investigation, that is, the experimenters were unaware of the formulations comprising each treatment group, which until the study was completed were only identified as Solution A, Solution B, etc.

The intent of this phase of the study was to determine whether the EpiThera®formulation or its components hastened the rate of superficial skin healing following partial thickness burns. Figure 1 shows composite photographs from a total of four rats: two different rats from each of the four treatment groups. At Day 7 after burn wounding, all wounds from all groups exhibited a statistically identical degree of inflammation and granulation tissue formation. On Day 14, wound healing had progressed in all groups (Figure 1), but Group C, receiving only EpiThera® without the carrier used in the formulation (i.e. without emu oil), exhibited significantly enhanced tissue inflammation, due to the presence of benzyl alcohol without the ameliorating effects of emu oil (see TB7-03 for discussion of the positive synergism of emu oil and benzyl alcohol).
By Day 28, a striking difference existed in the appearance of the wounds (Figure 1). The two populations that had been treated with either emu oil alone or with EpiThera® (Group A and B, respectively)had lost all eschars (scabs) and the nearly completely healed skin showed nearly complete epithelialization of the original burn site. However, 70% of Group C still exhibited eschars, while 42% of Group D still exhibited eschars. Figure 2 shows higher magnification photographs of the burn site of two rats, one treated with EpiThera® and one receiving no treatment.

Figure 1. Twenty eight day succession of superficial wound healing in rats receiving partial thickness burns. Rat identification number is indicated for each treatment group (left). Note the lack of eschars on Day 28 in rats receiving treatment with either emu oil or EpiThera® (red outline).

The purpose of this study was to investigate the effect of emu oil and EpiThera®, an emu oil-containing product, on the superficial appearance and rate of healing of partial-thickness skin burns in rats. Examination of the skin of rats over a 28 day time course for healing indicates that that treatment of the burn wound with either emu oil or EpiThera® resulted in a much more rapid restoration of the original appearance of the skin than treatment with formulation lacking emu oil or no treatment at all.

It is noteworthy that, while there was no significant difference between the effects of emu oil or EpiThera®, this study was conducted under as close to aseptic conditions as possible. The initial wounds were created on skin that had been sterilized with chlorhexidine solution, and animals were subsequently kept in laboratory conditions likely to be much cleaner than the conditions under which humans might typically recover from surgical or other wounds. Thus, in the absence of any major bacterial or fungal challenge, this study has indicated that either treatment containing emu oil appeared equally effective in accelerating the return of the superficial skin layers to their normal pre-burn appearance. However, EpiThera® is strongly anti-microbial (bacteria and yeast), due in part to the benzyl alcohol in the commercial formulation (Technical Bulletin TB11-04).

We predict that the identical rate of superficial healing produced by both treatment groups containing emu oil under laboratory conditions is unlikely to occur under significant infection challenge, where the anti-microbial properties of EpiThera® would probably lead to enhanced healing compared to emu oil alone. Future studies indicated the efficacy of EpiThera® should be directed at examining the interaction of wound healing and bacterial/fungal presence.

Hosgood, G. (2003) Wound repair and specific tissue response to injury. In: Textbook of Small Animal Surgery, D. Slatter (ed.). W. B. Saunders, Philadelphia, PA, p. 66-72.
Pavletic, M.M. (1999). Atlas of Small Animal Reconstructive Surgery. M.M. Pavletic (ed). W.B. Saunders, Philadelphia, PA, pp. 17, 30, 78.
Pope, E.R. (1993) Skin healing. In: Disease Mechanisms in Small Animals Surgery, M.J. Bojrab (ed.). Lea and Febinger, Philadelphia, PA, p. 151-153.
Yang, L., Chan, T., Demare, J., et al. (2001) Healing of burn wounds in transgenic mice over expressing transforming growth factor-1 in the epidermis. Am. J. Pathol. 2001;159:2147-2157.

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