StemWrap amniotic allografts accelerate wound healing

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Abstract from American College of Veterinary Surgeons Study Submission

Title: Amniotic allografts accelerate wound healing.

Fowler A1, Gilbertie J1, Prange T1, Barceló-Fimbres M2,  Werber B3, Ames BT,Schnabel LV1

AFFILIATIONS:

This study tested the hypothesis that the use of commercially available, preserved, equine amniotic allografts (eAM) would decrease distal limb wound healing time and improve healing quality compared to silicone and non-adherent dressings.

Two skin wounds were created on each metacarpus of 8 horses.  In each horse, one forelimb had one wound treated with eAM (StemWrap membrane and StemWrap+ injection); the other was subject to eAM injection effects (local StemWrap+). The opposite forelimb had one wound treated with a silicone dressing; the other was treated with a non-adherent dressing (negative control). Images were obtained twice-weekly for analysis of wound circumference (WC) and surface area (WSA). Each wound was normalized to initial treatment-day measurements. The study was continued until wounds healed, following which a biopsy sample was taken to evaluate healing quality.

Two silicone wounds and 1 negative control wound were grossly infected and non-healed; all remaining wounds were healed by day 59.  eAM and local StemWrap+ groups had significantly accelerated healing compared to the silicone group as determined by WC slopes (Linear regression; p=0.0042 and p=0.0008, respectively) and also by WSA slope for eAM (p=0.0440).  When mean healing rates were compared over the study, both eAM and local StemWrap+ groups had increased WC healing compared to silicone dressing and negative control groups (ANCOVA; p<0.05).

These results support the use of eAM for decreasing the healing time of equine distal limb wounds.  Healing quality assays are currently underway and will add additional information for future clinical application of these products.

Long Abstract

Title:  Commercially available acellular equine amniotic allografts accelerate healing of experimentally induced full-thickness distal limb wounds in horses compared to silicone dressings or non-adherent dressings.

Reasons for performing the study: Fresh amniotic membrane has been used for decades to treat equine wounds, but with conflicting results due to variable harvest procedures and problems with contamination.  Recently, commercially available amniotic products that can undergo sterile processing and manufacturing have been used in human medicine to treat wounds with repeated success.

Hypothesis/Objectives: This study tested the hypothesis that the use of commercially available acellular equine amniotic allografts (eAM, AniCell Biotech) would decrease healing time and improve the healing quality of equine distal limb wounds compared to silicone and non-adherent dressings.  The use of eAM, the combination of acellular dry equine amniotic membrane (StemWrap) and implantable acellular liquid morselized amniotic membrane (StemWrap+), was evaluated, as was the local effect of StemWrap+ injection.

Methods: Two full-thickness skin wounds were created on each metacarpus of 8 horses (Figure 1). In each horse, one randomly assigned forelimb had one wound treated with eAM; the other was subject to the effects of eAM injection and referred to as local StemWrap+Ò. The opposite forelimb had one wound treated with silicone dressing (CicaCare, Smith&Nephew); the other was treated with a non-adherent dressing (negative control).  All treatments were assigned equally to proximal and distal wounds.  Half-limb bandages were maintained and changed twice weekly.  At each bandage change, traditional and 3-dimensional images (Scanify, Fuel 3D) were obtained for serial analysis of wound circumference (WC) and surface area (WSA).  Excessive granulation tissue was debrided weekly for the first 4 weeks on all wounds. The study continued until wounds were healed, following which an 8mm biopsy punch sample was taken to evaluate quality of healing via histologic and immunohistochemistry assays to assess angiogenesis, apoptosis, and TGF-b1 levels.  Rate of wound healing was calculated as percentage decrease in WC and WSA over time, with each wound normalized to initial treatment day measurements. Slopes of WC and WSA curves between groups were analyzed by least squares linear regression models and measurements over the course of the study evaluated by ANCOVA (horse as covariant) with LSD post-hoc tests (Statistix 10, Analytical Software; p<0.05 considered significant).

Results Twenty-nine (29) of the 32 total wounds were healed by the conclusion of the study at 59 days.  All non-healed wounds (3) were grossly infected and were comprised of 2 silicone dressing treated wounds and 1 associated (on the same limb of that horse) negative control wound.  Due to the extent of the infection and treatment required, these wounds were not followed through to complete healing and the last measurement taken prior to fulminant infection was used for all remaining time points. eAM and local StemWrap+ treated wounds had significantly accelerated healing compared to silicone dressing treated wounds as determined by WC healing slopes (p=0.0042 and p=0.0008, respectively; Figure 2A) and also by WSA healing slope for eAM (p=0.0440; Figure 2B). When mean healing rates were compared over the course of the study, both eAM and local StemWrap+Ò groups had increased WC healing compared to silicone dressing and negative control groups (ANCOVA; p<0.05). A significant decrease in mean WC was found for eAM and local StemWrap+ groups on day 59 compared to the silicone dressing group (ANCOVA; p<0.05). Although all wounds initially increased slightly in WC as expected with no significant differences between groups (Figure 2C), the eAM group had significantly greater increases in WSA compared to the silicone dressing and negative control groups on day 7 due to greater granulation tissue formation (ANCOVA; p<0.05; Figure 2D). The local StemWrap+Ò group was statistically equivalent to all other groups at this time point. Following day 7, the granulation tissue production on eAM treated wounds decreased and was equivalent to the other groups. Quality of wound healing analyses are currently underway.

Discussion: This data supports the hypothesis that treatment with eAM decreases healing time of equine distal limb wounds.  The local effect StemWrap+ treatment led to a similar healing rate as treatment with eAM, but only eAM treatment led to increased production of granulation tissue early in the study.  This suggests that the dry amniotic membrane (StemWrap) is responsible for the formation of granulation tissue and should be used in wounds in which that is desirable such as deep wounds and wounds with exposed bone or soft tissue structures.  It was an unexpected finding that 2 of the silicone dressing treated wounds became infected, but it is theorized that the occlusive nature of the silicone dressing and its prolonged use facilitated such infection.

Main Study Limitations: Surgically created wounds do not accurately mimic the traumatic wounds on which these products are being used clinically. These wounds were treated promptly, and the silicone dressing was maintained for nearly 60 days, which may not reflect what would occur in a clinical setting.

Scientific or Clinical Relevance: This is the first study to evaluate the use of commercially available equine amniotic membrane products for wound healing. These products undergo rigorous quality control and are well packaged, easy to store in a hospital, and easy to transport for ambulatory work.  While the wound healing quality assays are currently underway, this study has shown that the use of eAM significantly decreases wound healing time in distal limb wounds. The results of this study also highlight the fact that product selection and timing of use is likely critical for future clinical application.  The combined dry and implantable (StemWrap and StemWrap+) products are mostly likely beneficial for deep wounds in which expedient production of large amounts of granulation tissue is desirable and may also be beneficial for chronic infected wounds.  StemWrap+ alone may be sufficient to improve the healing time of more superficial wounds.  Silicone dressings are typically used in horses to prevent the formation of exuberant granulation tissue, and therefore may be beneficial in the later stages of healing either alone or following treatment with eAM.

Figure 1.  Schematic of study design showing surgically created proximal and distal 6.25cm2 wounds on each forelimb.  The distal most aspect of each distal wound was at the level of the button of MC IV.  The distal most aspect of each proximal wound was 2cm proximal to the proximal most aspect of each distal wound.  Templates and rulers were used during wound creation to ensure consistent wound sizing.

Figure 2.  Wound healing measurement data calculated as percentage decrease in wound circumference (WC) and surface area (WSA) over time, with each wound normalized to initial treatment day measurements. Graphs for healing rates of WC (A) and WSA (B) are shown as scatter plots with slopes for each group. As shown by *, eAM and local StemWrap+ treated wounds had significantly accelerated healing compared to silicone dressing treated wounds as determined by WC healing slopes (p=0.0042 and p=0.0008, respectively; and also by WSA healing slope for eAM (p=0.0440). Graphs of healing data displayed as means ± SEM are shown for WC (C) and WSA (D).  The * shown for day 59 in (C) represents significantly improved healing (decreased WC) in the eAM and local StemWrap+ groups compared to the silicone dressing group and the * shown for day 7 in (D) represents the significant increase in WSA due to granulation tissue production observed for the eAM group compared to the silicone dressing and negative control groups (ANCOVA; p p<0.05; the local StemWrap+ group was statistically equivalent to all other groups at this time point.

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