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Biomechanics of Wound Healing in an Equine Limb Model

Biomechanics of Wound Healing in an Equine Limb Model: Effect of Location and Treatment with a Peptide-Modified Collagen–Chitosan Hydrogel

The equine distal limb wound healing model, characterized by delayed re-epithelialization and a fibroproliferative response to wounding similar to that observed in humans, is a valuable tool for the study of biomaterials poised for translation into both the veterinary and human medical markets. In the current study, they developed a novel method of biaxial biomechanical testing to assess the functional outcomes of healed wounds in a modified equine model and discovered significant functional and structural differences in both unwounded and injured skin at different locations on the distal limb that must be considered when using this model in future work. Namely, the medial skin was thicker and displayed earlier collagen engagement, medial wounds experienced a greater proportion of wound contraction during the closure, and proximal wounds produced significantly more exuberant granulation tissue. Using this new knowledge of the equine model of aberrant wound healing, they then investigated the effect of peptide-modified collagen–chitosan hydrogel on wound healing. Here, they found that a single treatment with the QHREDGS (glutamine–histidine–arginine–glutamic acid–aspartic acid–glycine–serine) peptide-modified hydrogel (Q-peptide hydrogel) resulted in a higher rate of wound closure and was able to modulate the biomechanical function toward a more compliant healed tissue without observable negative effects. Thus, they conclude that the use of a Q-peptide hydrogel provides a safe and effective means of improving the rate and quality of wound healing in a large animal model.

Distal limb wounds in horses are commonly encountered in clinical practice and are a challenge to treat. In this location, because of the frequency of extensive tissue loss and contamination, horses must often heal open wounds through second intention. Second intention healing relies on the formation of a granulation tissue (GT) to fill the wound bed, followed by restoration of the epithelial barrier through re-epithelialization and, to a lesser extent, wound contraction. On the distal limb of horses, this type of healing is plagued by a weak, yet extended inflammatory phase as well as by local ischemia and hypoxia. All these pathologies combined to contribute to the frequent development of exuberant granulation tissue (EGT), or “proud flesh”, which further delays wound healing by impeding both wound contraction and re-epithelialization.

A modified equine distal limb wound healing model and its biomechanical analysis.

(a) Modified orientation [two vertically arranged wounds dorsomedially (“medial”) and dorsolaterally (“lateral”) on both forelimbs] of wounds. E = no treatment, H = peptide-free hydrogel, QH = Q-peptide hydrogel, and RQH = Q-peptide hydrogel (repeated application). (b) Experimental timeline. Colored boxes represent relevant wound locations. (c) Prefabricated hydrogel bandages provided ease of application in a field setting. (d) Representative specimens of excised intact skin (day 0) and excised wounds (day 42) for mechanical testing. Markers placed on the tissue allowed for measurement of deformation. (e) Biaxial tensile device. Directions (11) and (22) are along and perpendicular to hair growth, respectively. Blue and red arrows indicate how forces are applied. (f–h) Derivation of effective mechanical properties from stress–deformation curves for the energy loss (EL) characterizing tissue viscoelasticity, the knee stretch (KS) indicating the stretch at which collagen fibers start to engage, and the elastic moduli (EM) identifying tangential stiffness in the fully engaged state. The respective stresses are plotted vs displacements of hooks in the (f) equidisplacement protocol, vs the central stretches based on markers’ tracking from (g) all the protocols, and vs the (h) interpolated equistretch protocol.

As a result, large wounds on the equine distal limb often result in poor functional and cosmetic outcomes as well as pose a significant economic burden to the owner. In a recent survey of practicing veterinarians, severe or poorly healing wounds ranked as the second most common cause of death or euthanasia in equine patients. Therefore, one of their key aims is to identify a therapeutic that can accelerate wound closure and improve the overall repair of wounds on the equine distal limb.

Wound location impacts the rate of closure and GT formation in the equine model.

(a) Thickness of the unwounded tissue from the medial and lateral skin. (b)Wound closure observations at different locations of both right and left limbs shown as a percentage closure from day 1. (c) Percent of wound closure occurring through re-epithelialization. (d) GT present in lateral wounds on day 14 upon removal of Tegaderm film in two horses. (e) GT scores of proximal wounds compared with those of distal wounds. * = p < 0.05, **** = p < 0.0001. n = 8.

Defective wound healing, ranging from chronic, nonhealing wounds to excessive fibroplasia as observed in hyperplastic scarring and keloids, is also a massive challenge in human medicine. A 2015 Global Burden of Disease study reported the prevalence of skin and subcutaneous disorders as affecting more than 600 million people; an increase of 22 % over the past 10 years. Skin diseases such as chronic, nonhealing wounds are only expected to continue to rise due to the increasing incidence of predisposing chronic diseases such as diabetes and the aging population. Already, the prevalence of chronic wounds has been estimated as 2.21 per 1000 people. Even when wound healing does occur, the resultant scar can develop a spectrum of clinical problems associated with poor dermal function often leading to chronic disability, altered sensation, and increased risk of reinjury. With an estimated annual economic burden of wound care in Canada topping $3.9 billion, the development of effective therapeutics aimed at overcoming deficient wound healing is of particular importance. However, before such therapeutics can reach clinical practice, it is imperative that safety and efficacy are confirmed in experimental models of wound healing.

Lack of preferential directions in biomechanical data and intrinsic difference between the unwounded skin and healing wounds.

Lack of preferential directions in biomechanical data and intrinsic difference between the unwounded skin and healing wounds. (a) KS ratios of each direction (11 and 22) for wounded and unwounded specimens. (b) Representative SHG images of collagen within the dermis in unwounded (day 0) and wounded (day 28) tissues. (c) Alignment of collagen fibers in the dermis, as observed on SHG images. (d) KS at day 0 and 42, medial compared with lateral. (e) KS and (f) EL in different limb locations expressed as a percent of that horses own for unwounded control from the same site. Comparison of (g) EM, (h) KS, and (i) EL in healing wounds with (“closed’) or without (“open”) a restored epithelial barrier. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. n = 8.

Rodents, though the most popular animal model of wound healing, heal primarily through wound contraction, thus differing greatly from that of humans who heal primarily through cellular proliferation and re-epithelialization.

The cellular processes underlying wound healing in horses more closely resemble those in humans. Indeed, human clinical conditions such as keloids and hypertrophic scarring are reminiscent of EGT observed in equine wound healing, as is the development of chronic, nonhealing wounds common among both species. Furthermore, the equine wound healing model has been demonstrated to produce only mild discomfort to subjects, and importantly, does not require euthanasia upon study completion, thus reducing and refining the use of animals in wound healing research. As such, distal limb wound healing in horses serves as a uniquely insightful preclinical (large animal) model that may provide a new understanding and new avenues for skin wound healing treatment in humans. Indeed, the treatment efficacy demonstrated in equine distal limbs would not only be of value in veterinary medical applications but also provide a strong impetus for a clinical trial in humans.

To address the challenges presented by complicated nonhealing wounds, biomaterials which seek to support the body’s natural wound healing process through the promotion of prohealing cues, epidermal cell migration and re-epithelialization, and immune system modulation have been a growing area of research.

A single treatment with a peptide-modified hydrogel improves the wound healing rate.

(a) representative gross wound images from horse #4 across 42 days. Note: hair loss around wound, if present, is the result of continuous contact observed with wound exudate underneath the bandage. (b) Survival curve indicating the time at which wounds were observed to be fully healed across treatment groups. (c) Percent of total wound closure measured as a function of a wounds own on day 1 control. (d) Granulation score (higher number indicates a worse appearance) of distal wounds on days 14, 28, and 42. (e) Percentage of wound closure occurring through re-epithelialization and contraction. Results reported as mean ± SD. n = 8. E = no treatment, H = peptide-free hydrogel, QH = Q-peptide hydrogel, RQH = Q-peptide hydrogel (repeated application). n = 8.

Recently, a novel hydrogel system has been developed, which incorporates an integrin-binding prosurvival peptide derived from angiopoietin-1, QHREDGS (glutamine–histidine–arginine–glutamic acid–aspartic acid–glycine–serine), into chitosan–collagen biocomposite material. This “Q-peptide hydrogel” (QH) has previously demonstrated efficacy in promoting keratinocyte attachment, survival, and migration, as well as the promotion of prohealing polarization of bone marrow-derived macrophages. The QH also demonstrated accelerated healing in a diabetic mouse model by promoting rapid re-epithelialization and improved physiological features. To further investigate the efficacy and safety of this product in a model that more closely recapitulates human wound healing, they have modified and further described an equine distal limb wound healing model by staggering wound location to evaluate multiple treatments and their associated controls in the same animal and tested the hypothesis that application of the QH will accelerate re-epithelialization, limit the development of hyperproliferative scar, and ultimately improve the biomechanical quality of the healed skin1.

Treatment with peptide-modified hydrogels shows no negative effects on the inflammatory response and re-vascularization during wound healing.

(a) Representative 20× images from day 14 biopsies stained for granulocytes and macrophages (MAC387—red) as well as for von Willebrand factor (vWF—green) to highlight endothelial cells within blood vessels. (b) Quantification of total MAC387+ immune cells per high-powered field. (c) Quantification of vWF+ blood vessels intersecting with a computerized grid overlaid per high-powered field. E = no treatment, H = peptide-free hydrogel, QH = Q-peptide hydrogel, and RQH = Q-peptide hydrogel (repeated application). Scale bars, 50 μM. Data represented as mean ± SD. n = 8.

Treatment with peptide-modified hydrogels does not alter the thickness of the healing scar during early healing (day 28). (a) Representative 20× images from day 28 biopsies cryosectioned (35 μm) and stained with H&E. (b) Epidermal and (c) dermal thickness measurements averaged across three separate locations within the healed wound edge (between dashed lines). (d) GT thickness measured in the area of open wound (*). E = no treatment, H = peptide-free hydrogel, QH = Q-peptide hydrogel, and RQH = Q-peptide hydrogel (repeated application). Data presented as mean ± SD. n = 8.

Effect of treatment on the biomechanical response during early wound healing.

(a) EM, (b) KS, and (c) EL for wounds with different treatments, shown as a percent change of its own on day 0. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. n = 8. E = no treatment, H = peptide-free hydrogel, QH = Q-peptide hydrogel, RQH = Q-peptide hydrogel (repeated application).

  1. Sparks, H. D. et al. (2021). Biomechanics of Wound Healing in an Equine Limb Model: Effect of Location and Treatment with a Peptide-Modified Collagen–Chitosan Hydrogel. ACS biomaterials science & engineering, 7 (1), s. 265–278. doi:10.1021/acsbiomaterials.0c01431

Biomechanics of Wound Healing in an Equi
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