CHAPTER 2 Jacintha M. Wilmink, DVM, PhD Observing differences in wound healing between horses and ponies has provided valuable information about the intrinsic process of wound healing and the common complications encountered when managing traumatic wounds in the equid. Ponies heal faster and with fewer complications than do horses. To a large extent, these differences can be explained by disparity in the local inflammatory response, which, in turn, relates to differences in the functional capacity of leukocytes. Research data indicate that a maximal effect of treatment will be obtained in clinical practice if a differential approach is used, optimizing conditions for each successive phase of wound healing. In particular, the effect of treatment on the inflammatory response is of paramount importance to the other phases of healing and should, therefore, always be considered when managing a wound. When treating wounds healing by second intention, inflammation should be stimulated until the wound has filled with granulation tissue, and thereafter it should be inhibited. The horse evolved over the course of millions of years from a small forest dweller to a large ungulate that inhabited the vast open plains of the temperate zone. It became a “flight” animal whose instinctive reaction to danger is to run.1 Evolution took place as a response to various environmental and climatic challenges, and the development of special features resulted from selection by mankind. Both evolution and selection led to the large variety of breeds known today. The equine species can be roughly subdivided into horses and ponies, and this division is determined by the height of an adult at the withers (ponies are <1.48 m). Whether ponies are just small horses has been debated for decades. The discussion of this topic, with respect to wound healing, started in the 1980s when Bertone et al.2 found, in a study examining second‐intention healing, that wounds of ponies heal faster than those of horses and without the formation of exuberant granulation tissue (EGT). Other authors, however, reported that wounds of ponies do develop EGT,3,4 and the faster healing of wounds in ponies could not be confirmed.4 Because a difference in wound healing between horses and ponies might provide information about the basic biology of equine wound healing and about the complications commonly associated with wound repair in this species, Wilmink et al. performed a series of experiments to investigate differences in wound healing between horses and ponies.5–7 They proved that skin wounds in ponies heal faster and with fewer complications than do similar wounds in horses and demonstrated that these differences were based on the efficiency and capacity of the leukocyte to produce various mediators.8,9 Differences in healing are, however, apparently not related to body size. This is confirmed by a study in donkeys and Caspian miniature horses, equine subspecies of similar size, which showed that wound healing in donkeys is faster than healing in Caspian miniature horses.10 What remains to be elucidated is the cause of the observed differences in healing between horses and ponies. The longer period of domestication of the horse may have precluded natural selection against poor healing, because the wounded horse was/is tended to by man. Additionally, the artificial selection of various features, such as height, athletic capacity, and appearance, might have favored the development of some diseases and undesirable characteristics, including the reduced efficiency of leukocytes. Moreover, horses with wounds so poorly healed that athletic ability is impaired are often used for breeding, thus perpetuating a hereditary tendency toward poor healing. In contrast, ponies have been domesticated for a much shorter time, and, consequently, a poor capacity for healing may have been eliminated by natural selection. Moreover, artificial selection within pony breeds has been less intense, and many pony breeds maintain subpopulations (and genetic reserves) in the wild. The hypothesis that natural and artificial selection have an influence on wound healing is supported by the faster healing of donkeys compared to Caspian miniature horses,10 the latter being selected based on a special feature, namely their speed. The physiologic and pathophysiologic differences in healing observed between horses and ponies with experimentally induced wounds have since been corroborated by other research7–9,11,12 and by clinical observation. These observations have resulted in improvements in guiding the management of wounds of equids and led to faster healing and the prevention of complications. Primary closure of traumatic wounds of horses is preferred over healing by second intention because healing by primary intention is usually faster, and the cosmetic and functional results are better. Unfortunately, either partial or complete wound dehiscence may ensue after primary closure. Whether or not a wound dehisces depends on many factors, including those relating to the wound itself, to the horse, to the treatment, and to the environment.13,14 The success of primary closure of traumatic wounds was evaluated retrospectively by Wilmink et al. in more than 500 equine patients admitted to a referral clinic.7 The study revealed that primary closure is significantly more often successful in ponies than in horses (Figure 2.1), and that bone sequestra form significantly less often in ponies when bone is exposed during injury. The predominant cause of wound dehiscence and of sequestration of bone is infection.7,16,17 Other factors contributing to dehiscence are tension on the wound’s margins, excessive movement of the sutured region, and involvement of certain structures, such as tendons, ligaments, and synovial cavities. Other factors besides infection contributing to formation of a sequestrum include exposure of cortical bone and extensive trauma.18 The risk of infection is influenced by many factors, including the time elapsed since injury, the degree of contamination, the degree of tissue damage, and the thoroughness of debridement and irrigation. Wound debridement determines the concentration of bacteria left in the wound and is one factor that can be controlled clinically.13,14,17 The concentration of bacteria, in combination with factors at the wound that facilitate bacterial colonization, such as the presence of dead space, a hematoma, and devitalized tissue, is critical to whether or not a wound becomes infected (the reader is referred to Chapter 4 for more information about factors involved in the development of infection). Although bacterial colonization and development of infection are greatly influenced by the administration of antibiotics, the effectiveness of the patient’s own local defense, its acute inflammatory response, is as least as important to the prevention of infection.19 Figure 2.1 (a) Wound on the elbow of a horse, which suffered dehiscence following primary closure, as a result of infection. (b) Wound on the distal aspect of the limb of a 4‐year‐old pony with an open fetlock joint and damage to the lateral collateral ligament. The wound was sutured approximately 8 hours after it occurred and it healed successfully, without dehiscence. Source: (b) Wilmink 2004.15 Reproduced with permission of Elsevier. In the aforementioned retrospective study by Wilmink et al., all these factors relating to wound infection were evaluated and compared between horses and ponies.7 Ponies and horses did not differ with respect to age and gender, nor with respect to wound characteristics, such as location, duration, contamination, and treatment. The wounds of ponies, however, were generally deeper, and ponies were more apt to suffer laceration of an extensor tendon, to incur damage to the periosteum, to have exposed bone, and to have their wound closed while standing, rather than while anesthetized. Additionally, ponies were significantly less likely to receive antimicrobial therapy and to be administered a non‐steroidal anti‐inflammatory drug (NSAID). Ponies were, therefore, at greater risk of bacterial challenge than were horses because their wounds were deeper and debridement was less complete since it was performed with the ponies standing, and because they were less likely to receive antimicrobial therapy. In spite of this greater risk, wound infection occurred less often, implying that the acute inflammatory response in ponies is more effective than that of horses at reducing bacterial contamination. The more frequent use of NSAIDs in horses, however, may have contributed to a reduced effectiveness of the inflammatory response. Wounds are allowed to heal by second intention when closure is not feasible or affordable, or when a wound dehisces after being closed primarily. Second‐intention healing was investigated in horses and ponies by creating excisional wounds on the metatarsi and buttocks that extended through the periosteum of the metatarsal bone or 18 mm into the muscle on the buttocks, in imitation of clinical wounds.5,6 This investigation found that wounds of ponies heal significantly faster than do wounds of horses (Figure 2.2) and that the speed and efficiency of healing seem related to remarkable differences between horses and ponies in the phases of healing.5,6 Subsequent studies showed that these differences can be attributed to variations in the function of leukocytes.8,9,11 Figure 2.2 Wound area as a function of time (mean + s.e.m.). HMT, metatarsal wounds of horses; HB, body wounds of horses; PMT, metatarsal wounds of ponies; PB, body wounds of ponies. Source: Wilmink 1999.5 Reproduced with permission of John Wiley & Sons. Wound healing is often divided into general phases of hemostasis, inflammation, proliferation, and synthesis and remodeling of matrix (see Chapter 1). Because these phases overlap and occur simultaneously in all tissue components, distinguishing them from one another is difficult. Consequently, this division is somewhat academic. In a clinical setting, dividing healing into the following macroscopically apparent events may be more practical: inflammation, formation of granulation tissue, wound contraction, and epithelialization. Although these events also overlap, they largely succeed one another and occur more or less chronologically. Moreover, they are clearly visible to the practitioner observing second‐intention healing. The inflammatory response to wounding is more prompt in ponies, as demonstrated by comparing the leukocytic infiltration of experimental wounds of ponies and horses.6 High numbers of polymorphonuclear cells (PMNs) are found during the first 3 weeks of healing, after which the PMNs disappear rapidly from the wounds of ponies. In contrast, the influx of PMNs is sluggish and weak in horses, but PMNs persist so that, beginning 4 weeks after wounding, the number of PMNs exceeds those in the wounds of ponies.6 The leukocytes of ponies are more efficient than those of horses. An in vitro study showed that the leukocytes of ponies produce more reactive oxygen species, such as H2O2 or O− radicals, necessary for bacterial killing after phagocytosis.9 Within tissue cages implanted subcutaneously and in newly formed granulation tissue, the leukocytes of ponies also produce higher concentrations of other inflammatory mediators, including tumor necrosis factor (TNF)‐α, interleukin (IL)‐1, and transforming growth factor (TGF)‐β, essential to the reinforcement of the inflammatory response, to the induction of fibroplasia, and to wound contraction.8,9 The superior production of these mediators can thus explain the higher initial influx of leukocytes into wounds of ponies. Migrated leukocytes, in turn, release more biologically active substances, thus creating a positive feedback that enhances the inflammatory response.19,20 This feedback may account for the faster debridement of non‐viable tissue and fibrin in wounds of ponies, leading to the faster development of a healthy bed of granulation tissue. In contrast, granulation tissue in the wounds of horses retains an irregular and purulent appearance, along with persistent deposits of fibrin.5,6 An enhanced inflammatory response observed in ponies can, likewise, translate into a more efficient local defense against contaminating bacteria, leading to a superior control of wound infection.7 A stronger acute inflammatory response, thus, averts the development of chronic, low‐grade inflammation and leads to a faster and more thorough preparation of the wound for repair. Indeed, chronic inflammation, such as that seen in horses,6 perpetuates the release of tissue‐damaging lysosomal enzymes, as well as mediators, such as TGF‐β, which in turn, over‐stimulate fibroplasia and lead to the formation of EGT, which inhibits contraction.19,21–23 In summary, the inflammatory response to wounding in ponies is stronger and more succinct; this response appears to be more efficient for healing. In contrast, the inflammatory response in horses is weak in onset but persists over time, and this poor but persistent response may be due to the lower initial production of inflammatory mediators. In a study by Wilmink et al., granulation tissue formed faster in experimental wounds of horses than in those of ponies.5 The abundant granulation tissue in the wounds of the limbs of horses was observed to push the edges of the wound apart, which may explain why experimental wounds on the limbs of horses enlarged so dramatically during the first 2 weeks after they were created (Figure 2.2). Between weeks 2 and 3 of healing, the granulation tissue of all wounds on the limbs and buttocks of horses and ponies protruded above the level of the surrounding skin and was characterized as excessive (clinically referred to as EGT or “proud flesh”); protrusion of granulation tissue was most prominent in wounds on the limbs of horses (Figure 2.3). The wounds were left unbandaged after week 3, and during week 4 EGT disappeared spontaneously from wounds, except those on the limbs of horses, where the excess tissue had to be trimmed to promote healing.5 The granulation tissue in horses was traversed by grooves and clefts for a much longer period, and presented a yellowish purulent surface up to week 5 after wounding. The appearance of the granulation tissue on the limbs may have been due to the weak and delayed onset followed by prolongation of the inflammatory phase. In contrast, the granulation tissue in the wounds of the ponies was smooth, regular, and achieved a healthy pink color significantly sooner (Figure 2.4).5 It was microscopically apparent that fibroblasts continued to proliferate in horse wounds even after granulation tissue filled the wound bed, whereas fibroblasts in pony wounds ceased proliferating at this time. Granulation tissue in wounds of horses presented a chaotic cellular pattern and appeared persistently inflamed, whereas it was organized regularly in pony wounds (Figure 2.5).6 Figure 2.3 After 3 weeks, the limb wounds of horses (a) contained more exuberant granulation tissue than the limb wounds of ponies (b). In the same timeframe, the wounds on the buttocks of horses (c) and ponies (d) had formed some exuberant granulation tissue protruding above all wound margins. Source: (a and b) Wilmink 2004.15 Reproduced with permission of Elsevier. Figure 2.4 (a) By 4 weeks after wounding, the granulation tissue of the limb wounds of horses was traversed by grooves and clefts, and on its surface was a purulent exudate. (b) The granulation tissue of pony wounds, on the other hand, was smooth, regular, and had a healthy pink color. Source: Wilmink 2004.15 Reproduced with permission of Elsevier. Figure 2.5 Typical microscopic appearance of the granulation tissue of metatarsal wounds 6 weeks after wounding (DAP‐filter, MIB). (a) In horses, many brown‐colored cells, actively synthesizing DNA and preparing for mitosis, were present. Note the irregular arrangement of the fibroblasts in the tissue and the presence of polymorphonuclear leukocytes. (b) In ponies, only a few brown‐stained cells are seen in the regularly organized granulation tissue. Source: Wilmink 1999.6 Reproduced with permission of John Wiley & Sons. There may be a causal relationship between persistent inflammation and the continuous proliferation of fibroblasts and the synthesis of granulation tissue, via the activity of mediators, such as TNFα, IL‐1, IL‐6, platelet‐derived growth factor (PDGF), TGF‐β, and basic fibroblast growth factor (bFGF), which are known to induce fibrosis.22–24 However, the formation of granulation tissue was less extensive in the wounds of ponies than in those of horses, particularly those located on the limb, even though the granulation tissue of ponies initially contained higher concentrations of TNFα, IL‐1, and TGF‐β,5,8,9 which mediate the migration and proliferation of fibroblasts and endothelial cells.22 Furthermore, fibroblasts from limbs of ponies are known to proliferate faster in vitro than those of horses,11,25 even though fibroplasia seems more rapid, in vivo, in horses. Apparently, the balance of mediators, the interaction of mediators with other factors, and time‐scale of the presence of mediators in vivo are more important than the absolute concentration of mediators in determining the rate of cellular growth, stressing once more the significance of the overall course of the inflammatory response. The formation of granulation tissue in horses is excessively fast, compared to that of other species26 and to what is observed in ponies.5 The fast formation and persistent proliferation of granulation tissue, caused by an unrelenting inflammatory response, clearly contribute to the formation of EGT.5,6 Contraction of wounds of ponies is faster and significantly more pronounced than is contraction of wounds of horses, and contraction of wounds on the body is faster and significantly more pronounced than is contraction of wounds on the limb (Figure 2.6).5 As a result, second‐intention healing is significantly faster in ponies than in horses and significantly faster in wounds on the body than in wounds on the limb (Figure 2.2).5 Figure 2.6 After 9 weeks of healing, the contribution of contraction to wound closure can be appreciated by observing the tattoo patterns close to the original margin of the wound. (a) The metatarsal wounds of horses have decreased in size. The tattoos show that the wounds have contracted minimally, whereas pronounced epithelialization is visible. (b) Closure of the metatarsal wounds of ponies was, to a large extent, the result of contraction and some epithelialization. (c) The buttock wounds of horses also healed mainly by contraction, with a small amount of epithelialization. (d) The buttock wounds of ponies healed, mainly by contraction, with very little epithelialization. The scar of this pony was unfortunately superficially damaged while shaving the hairs around the scar for the photographs. Source: (a and b) Wilmink 2004.15 Reproduced with permission of Elsevier. Histologic examination of wounds showed that the myofibroblasts in newly formed granulation tissue of wounds of ponies are organized into a regular pattern within 2 weeks after wounding; the cells are oriented perpendicular to the vessels and parallel to the wound’s surface. This pattern is thought to enhance contraction because a good bond between fibroblasts and the surrounding ECM is required for the contractile forces exerted by smooth muscle actin filaments within the fibroblasts to pull the margin of the wound centripetally (i.e., towards the center of the wound).27,28 Although the number of fibroblasts and the amounts of smooth muscle actin and collagen do not differ between wounds of horses and those of ponies, the alignment of myofibroblasts of horses is delayed (Figure 2.5).6 Wound contraction occurs when the forces exerted by myofibroblasts exceed centrifugal (i.e., outward) forces and the local resistance to displacement. Centrifugal forces present in the skin of horses and ponies are similar, as evidenced by the identical enlargement that occurs immediately after the creation of experimental wounds (Figure 2.2). Moreover, there is no reason to believe that the local resistance to contraction in horses and ponies should differ. Variations in wound contraction are, therefore, most likely related to differences in the contractile force generated by myofibroblasts within the granulation tissue. Surprisingly, the inherent capacity of fibroblasts of ponies to contract, in vitro, is similar to that of horses,11 suggesting that factors in the wound, such as the presence of inflammatory mediators, determine the contractile forces exerted by myofibroblasts and hence the extent of contraction. Indeed, inflammatory mediators, in particular TGF‐β, wield major effects on contraction. Interestingly, the concentration of TGF‐β is significantly higher in the newly formed granulation tissue of experimental wounds of ponies.8 This may explain the faster organization of myofibroblasts and the more extensive contraction observed in wounds of ponies because TGF‐β stimulates the differentiation of fibroblasts into myofibroblasts;29 induces α‐smooth muscle actin, α1β1 and α1β2 integrins, collagen, and fibronectin, all factors necessary to wound contraction;30 and enhances contractile forces.31 Furthermore, because other inflammatory mediators, such as prostaglandin (PG)E1, PGE2, TNFα, IL‐1, IL‐6, and interferon (IFN)‐γ inhibit contractility,32 a chronic inflammatory response, characteristic of wounds of horses, may exacerbate the deficiency of contraction noted in wounds of horses. In summary, wounds of ponies heal faster by second intention than do wounds of horses because contraction contributes more to closure of wounds of ponies than to wounds of horses (Figures 2.2 and 2.6). The differences in contraction are not caused by disparity in the innate contractile capacity of the fibroblasts but by the balance of mediators in the wound’s environment. The poorer contribution of contraction to closure of wounds on the limb of horses can be attributed to the initial low production of TGF‐β and the frequent occurrence of chronic inflammation.5,6,8 Proliferation of epithelial cells in experimental wounds of horses was similar to that of epithelial cells in experimental wound of ponies during the first weeks of healing. Cellular proliferation was temporarily reduced when EGT developed at 3 weeks in wounds on the limbs and the buttocks of horses and ponies.6 After the third week of healing, an inverse correlation developed between the area of epithelialization and wound contraction, so that wounds contracting the most epithelialized the least (pony versus horse wounds, buttock versus limb wounds) (Figure 2.6, Figure 2.7).5 This inverse correlation likely developed because the circumference of the wound margin furnishing migrating and proliferating epithelial cells decreased. Thus, more epithelialization was seen when wound contraction was poor, such as occurred in wounds on the limb of horses (Figure 2.6).5 Epithelialization of these poorly contracting wounds was significantly faster than was epithelialization of rapidly contracting wounds from 6 weeks onward, resulting in the largest area of newly formed, inferior‐quality epithelium and the most pronounced scars (Figure 2.8).5 Figure 2.7 Relative contribution of contraction and epithelialization to wound closure. For abbreviations see Figure 2.2. The correlation between the epithelialized area and wound contraction is inversed, so that wounds demonstrating the most contraction show the least amount of epithelialization (pony versus horse wounds, body versus limb wounds). Source: Wilmink 1999.5 Reproduced with permission of John Wiley & Sons. Figure 2.8 Scar of a metatarsal wound of a horse (a) and a buttock wound of a pony (b) 1.5 years after healing. The limb wound in the horse healed mainly by epithelialization, leaving an unsightly scar. The buttock wound of the pony closed mainly by wound contraction, leaving no visible scar. Source: Wilmink 2004.15 Reproduced with permission of Elsevier. Epithelialization is also hindered by persistent inflammation, which promotes release of toxic products and lysosomal enzymes from leukocytes. These toxic products not only damage tissue but also inhibit epithelial mitosis by altering the critical balance of cytokines and growth factors upon which epithelial cells depend.33 Additionally, epithelial mitosis appears to be negatively influenced by the presence of EGT and/or factors inducing EGT,34 as suggested by the finding that the mitotic activity of epithelial cells in all wounds temporarily diminishes when EGT is present.6 In other words, epithelialization slows when contraction is rapid, as seen in ponies. An increased rate of epithelialization, as seen in wounds on the limb of horses, however, has little effect on the speed of healing because epithelialization is inherently slow. A more extensive scar, characterized by fragility, poor attachment to the underlying basement membrane, and the absence of skin appendages, is formed when epithelialization is the primary mode of wound closure (Figure 2.8).35 Traumatic wounds of ponies are more apt to heal with a favorable cosmetic and functional outcome than are similar wounds of horses. This usually translates into lower costs of treatment for ponies than for horses. A pony’s better prognosis for favorable healing may justify treatment even when a wounded pony has such a low economic value that the owner is reluctant to treat the pony. The results of the study by Wilmink et al. examining primary‐intention healing and formation of sequestra in horses and ponies7 illustrate the detrimental effect of infection in the process of healing and the substantial role of the inflammatory response in local defense. As yet, there are no proven ways to stimulate the inflammatory response in sutured wounds to improve local defense mechanisms, signifying that preventing infection is of paramount importance, particularly for horses. To prevent infection, as many bacteria as possible should be removed by debridement and irrigation, and, therefore, because surgical debridement is more difficult when the patient is standing, general anesthesia should be the preferred method of restraint, especially when the wound to be debrided is extensive. Debridement of exposed cortical bone appears to protect the bone from infection and subsequent formation of a bone sequestrum. Proliferation and invasion of bacteria remaining after debridement can be prevented by appropriate antimicrobial prophylaxis. Equids suffering from an acute extensive traumatic wound should be administered a broad‐spectrum antibiotic as soon as possible after wounding, even if the patient is to be referred to a hospital better equipped to treat it. Antimicrobial therapy should be administered intravenously to ensure an immediate and adequate concentration of the antimicrobial drug within the wounded tissue (the reader is referred to Chapter 19 for more information about antibiotic therapy). Sutured wounds on a limb should be protected with a bandage or cast to reduce edema and to increase the local temperature, thereby facilitating perfusion of the wound with an adequate supply of leukocytes and oxygen. Bandaging also stimulates biologic processes, such as the production of endothelial and epithelial cells and fibroblasts as well as the synthesis of components of the extracellular matrix (the reader is referred to Chapter 6 for more information about the products used to dress and bandage wounds and Chapter 7 for information about the techniques of bandaging, splinting, and casting). The fact that the inflammatory response should initially be stimulated, rather than inhibited, implies that the wounded equid should not be administered a corticosteroid, topically or systemically. Furthermore, the routine use of NSAIDs should be avoided, because NSAIDs have been reported to exert adverse effects on wound healing.36–40 High doses and prolonged use of a NSAID have been shown to reduce inflammation in surgical wounds of horses and ponies;36,37 consequently, administration of a NSAID may increase the likelihood of infection in a traumatic wound. Additionally, certain NSAIDs have been shown to exert an adverse effect on skin flap survival.39 Administration of a NSAID may be warranted, however, if the equid is severely lame or if the wounded region is so swollen that local circulation could be compromised. In these instances, a NSAID should be administered, but at the lowest effective dose and for as short a duration as possible. Topical use of disinfectants and antibiotics should also be avoided as many products are toxic to wound cells or inhibit leukocyte function.41 Similarly, local anesthetics should not be infiltrated close to the wound, as these agents are also toxic for leukocytes and reduce their mobility and metabolism.42–44 Consequently, regional (perineural) anesthesia or a line block performed distant to the wound is preferred.13 In conclusion, measures should be taken to reduce contamination and to minimize the detrimental effects of treatment on the normal inflammatory response, particularly when the wounded equid is a horse.
Differences in Wound Healing between Horses and Ponies
Summary
Horses and ponies: same species, different healing characteristics
First‐intention healing (primary wound closure)
Second‐intention healing
Clinically apparent phases during wound healing
Inflammation
Formation of granulation tissue
Wound contraction
Epithelialization
Clinical application of the results of research
First‐intention healing (primary wound closure)
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