Limb Wound Healing Research Articles

The modified Meek technique as a novel method for skin grafting in horses: evaluation of acceptance, wound contraction and closure in chronic wounds

The acceptance of skin grafts in horses is unpredictable and the final cosmetic result can be disappointing. Besides movement and infection, graft failure is often caused by chronic inflammation, inherently present during second intention healing of limb wounds in horses. In human burns affected by infection and inflammation, the acceptance of the island skin grafts of the modified Meek technique appeared to be better than meshed sheet skin grafts. The percentage take of Meek micrografts is higher than of other techniques; and rates of both wound contraction and epithelialisation are increased. Large traumatic limb wounds of 13 horses healing by second intention were grafted using the modified Meek technique. Photographs of the wounds were taken at set intervals. Wound areas, and areas of acceptance and rejection were determined using a digital image post processor (Scion Image). The percentages of take, wound contraction and epithelialisation were calculated. The initial mean wound area was 7500 mm2. Graft acceptance was mean +/- s.d. 93.7 +/- 5.9%. Wound closure was due to contraction (55.2 +/- 11.1%) and epithelialisation (44.8 +/- 11.1%) and resulted in a 96.7 +/- 3.6% reduction of the initial wound area 29.1 +/- 6 days after grafting. All wounds showed functional and cosmetic healing. The method for skin grafting in horses achieved higher percentages of take than reported previously and consistent cosmetic and functional results. The grafts increased not only the rate of epithelialisation but also had a strong positive effect on wound contraction, resulting in rapid closure and smaller scars. The modified Meek technique proved to be a novel technique for skin grafting equine wounds in clinical practice, which can be performed easily. The molecular background of the increase of wound contraction by the grafts may provide a clue in the search for medicinal stimulation of wound contraction during second intention healing.

Application of Exogenous Esterified Hyaluronan to Equine Distal Limb Wounds

The objective of this study was to evaluate the efficacy of topical application of a hyaluronan (HA) derivative in wound healing with respect to the rate of epithelialization, fibroplasia, angiogenesis and contraction, magnitude of the local inflammatory response, local expression of transforming growth factor-β 1 and 3 (TGF-β 1 and 3), tumor necrosis factor-α (TNF-α), and collagen type III deposition. In six healthy adult horses, six full-thickness skin wounds were created on the dorsal aspect of both metacarpi using a sterile template. Sites were sampled at 0, 1, 2, 5, 14, 21, and 35 days following wounding. Wounds on one limb were dressed with commercially available esterified HA fleece under a nonadherent dressing. The opposite limb was covered with the nonadherent dressing alone (control). Images of the most proximal wounds were used to determine the area of total healing and the relative contributions of epithelialization and contraction to healing. At each sample time, a control and treatment biopsy were taken for histological evaluation and special stains. All samples were evaluated for degree of inflammation, fibroplasia and angiogenesis; in situ hybridization for type III collagen, TGFβ1 and 3, and immunohistochemistry for TNF-α. Mean percentages of total wound healing, epithelialization, and wound contraction were not significantly different between control and treatment groups. In treated horses, initial wound expansion was significantly decreased during the first 2 weeks. Mononuclear cell numbers, counted in the granulation tissue, increased in both control and treated limbs over the entire course of the study. However at day 35 the macrophage numbers counted in the treated horses were significantly increased compared with the control limbs ( P < .05). Although not statistically significant, relative staining for type III collagen in the treated wounds was less than that of control wounds. Results of the present study do not support a benefit of an exogenous HA-derivative in the healing of distal limb wounds in horses. The shortcomings of the study design are discussed.

Picroliv Accelerates Epithelialization and Angiogenesis in Rat Wounds

Tissue repair and wound healing are complex processes that involve inflammation, granulation and tissue remodeling. Angiogenesis plays a central role in wound healing. Earlier, we have shown that picroliv, a natural product obtained from the roots of Picrorhiza kurrooa, up-regulates the expression of vascular endothelial growth factor in human umbilical vein endothelial cells and of insulin-like growth factor in rats during hypoxia. In the present study, we have investigated the effect of picroliv in an ex vivo rat aorta ring model of angiogenesis. Picroliv enhanced the sprouting and migration of endothelial cells. We also investigated punch wound healing on days 4 and 7 after wounding by histology, morphometry and collagenization. The data showed improved re-epithelialization, neovascularization and migration of various cells such as endothelial, dermal myofibroblasts and fibroblasts into the wound bed after picroliv treatment. Immunohistochemical localization showed increased VEGF and alpha smooth muscle actin staining consistent with an increased number of microvessels in granulation tissue. These findings suggest that picroliv could be developed as a therapeutic angiogenic agent for the restoration of the blood supply in diseases involving inadequate blood supply such as limb ischemia, ischemic myocardium and wound healing.

Endothelial Progenitor Cell Release into Circulation Is Triggered by Hyperoxia‐Induced Increases in Bone Marrow Nitric Oxide

Endothelial progenitor cells (EPC) are known to contribute to wound healing, but the physiologic triggers for their mobilization are often insufficient to induce complete wound healing in the presence of severe ischemia. EPC trafficking is known to be regulated by hypoxic gradients and induced by vascular endothelial growth factor-mediated increases in bone marrow nitric oxide (NO). Hyperbaric oxygen (HBO) enhances wound healing, although the mechanisms for its therapeutic effects are incompletely understood. It is known that HBO increases nitric oxide levels in perivascular tissues via stimulation of nitric oxide synthase (NOS). Here we show that HBO increases bone marrow NO in vivo thereby increasing release of EPC into circulation. These effects are inhibited by pretreatment with the NOS inhibitor l-nitroarginine methyl ester (l-NAME). HBO-mediated mobilization of EPC is associated with increased lower limb spontaneous circulatory recovery after femoral ligation and enhanced closure of ischemic wounds, and these effects on limb perfusion and wound healing are also inhibited by l-NAME pretreatment. These data show that EPC mobilization into circulation is triggered by hyperoxia through induction of bone marrow NO with resulting enhancement in ischemic limb perfusion and wound healing.

Adult vascular progenitor cells and tissue regeneration in metabolic syndrome

Adult vascular progenitor cells (for example endothelial progenitor cells, EPC) have been studied for their contribution to vascular repair and angiogenesis. These cells can differentiate from bone marrow cells as well as circulating cells carrying hematopoetic stem cell markers. In vivo, they take part in vasculogenesis in different animal models of limb ischemia, myocardial infarction and wound healing. In metabolic disease, the outgrowth and function of EPC in vitro is defective and numbers of EPC correlate with classical risk factors of cardiovascular disease suggesting a role of EPC in the development of vascular complications. Pilot studies for the treatment of myocardial infarction and limb ischemia with autologous bone marrow showed a distinct therapeutic benefit that is presumably mediated by vasculogenesis in damaged tissues. However, little is known about the nature of EPC and their capability to differentiate into functional cells for tissue regeneration. In this article, we review and discuss the hitherto identified physiological function of EPC, the mechanisms leading to dysfunction of these cells and potential therapeutic applications in patients with metabolic syndrome or diabetes mellitus and vascular complications.

Autologous Peripheral Blood Stem Cell Implantation as Therapeutic Angiogenesis for Severe Limb Ischemia Autologous Peripheral Blood Stem Cell Implantation as Therapeutic Angiogenesis for Severe Limb Ischemia.

It is estimated that 100 million individuals suffer from severe limb ischemia worldwide. Here, we report a novel therapy for severe limb ischemia by transplanting autologous peripheral blood stem cells (PBSCs). Thirty patients with different limb ischemia were enrolled and randomized to either stem cell injection or standard therapy in the study. The patients in the transplant group received intramuscular injections of autologous PBSCs collected after G-CSF-induced stem cell mobilization. Lower limb pain, ulcer repair and wound healing and blood perfusion were improved in the cell treated group. All cell treated patients achieved successful limb salvage. Analysis by digital subtraction angiography demonstrated the formation of new collateral vessels in the ischemic limbs of transplanted patients after cell injection. In contrast, no significant improvement was observed in the control patients. These results provide pilot evidence indicating that transplantation of autologous PBSCs is a simple and effective therapeutic angiogenesis for limb ischemia. To determine the mechanism of action, several animal experiments were performed by transplanting human PBSCs via tail vein into nude mice. It was found that the transplanted cells survived and incorporated into capillary networks in murine ischemic limb. Laser Doppler Perfusion Image (LDPI) and histological analysis revealed that the transplantation of human PBSCs augmented blood flow and neovascularization in the ischemic hindlimb, ameliorated necrosis and improved limb salvage. Our results demonstrate that PBSCs are excellent cell population capable of augmenting neovascularization.

Wound repair in the horse: Problems and proposed innovative solutions

Second intention healing of full-thickness limb wounds in the horse is subject to numerous complications. In particular, an inefficient but protracted inflammatory response may preclude the ensuing proliferative and remodeling phases of the repair process from proceeding normally. Furthermore, the horse seems afflicted with an excessive fibroblastic response leading to the exuberant formation of granulation tissue and subsequent retardation of epithelialization and wound contraction. This article will discuss recent discoveries pertaining to the cellular and molecular mechanisms regulating wound repair in the horse. It will then address innovative solutions that have been implemented in the equine species. Although conservative and time-honored methods of wound care are unlikely to disappear, the development of substances that stimulate repair are imminent and equine practitioners must remain aware of these innovations to better serve their clients and patients.

Treatment of exuberant granulation tissue

The formation of exuberant granulation tissue almost inevitably occurs during second-intention healing of limb wounds in horses. It can be described as an aberrant prolongation of the phase of granulation tissue formation, rather than the wound making a transition to contraction. The formation of exuberant granulation tissue is affected by several factors such as bandages, casts, chronic inflammation, wound location and motion, tissues involved, breed and iatrogenic factors. Uncommonly a neoplastic transformation may occur. Among all the factors, the presence of chronic inflammation may be the most important and often not recognized cause for exuberant granulation tissue. Treatment depends on the age of the exuberant granulation tissue and the cause. The cause should be identified and treated. Many agents have been used to treat or prevent exuberant granulation tissue; however, caustic agents or cryogenic surgery should not be used, because they prolong chronic inflammation and cause more proliferation. In general, the best therapy is surgical excision of the protruding tissue and several excisions may be required. When the chronic inflammation subsides, the wound will resume healing by contraction and epithelialization.

Infrapopliteal angioplasty

Endovascular therapy for infrapopliteal vascular disease is gaining acceptance as there is growing evidence demonstrating its safety and effectiveness. It is most commonly used to treat patients with chronic critical limb ischemia (CLI) for limb salvage and wound healing. Its use to treat lifestyle altering claudication remains controversial. For the treatment of CLI, limb salvage rates with infrapopliteal percutaneous transluminal angioplasty (PTA) are high enough that these techniques are offering an alternative to bypass surgery. Current patency rates from infrapopliteal PTA can be improved further by proper patient selection, ensuring straight-line flow to the foot in at least one tibial vessel, and close patient surveillance for early reintervention. Possible future advances including the use of drug-eluting stents, cutting balloons, and aggressive antiplatelet regimens are being tested to improve clinical outcomes following endovascular interventions on the tibial arteries.

Biological activities of fibroblast growth factor-2 in the adult myocardium.

Fibroblast growth factor-2 (FGF-2) is a potent regulator of many cellular functions and phenomena, including cell proliferation, differentiation, survival, adhesion, migration, motility and apoptosis, and processes such as limb formation, wound healing, tumorigenesis, angiogenesis, vasculogenesis and blood vessel remodeling. In the adult myocardium, FGF-2 is expressed by various cell types, including cardiomyocytes, fibroblasts and smooth muscle cells. The biological effects of FGF-2 in the myocardium are mediated by the high-affinity tyrosine kinase receptor FGFR-1, the major FGF receptor in the heart. Here, we give an overview of current insights into the multiple roles of FGF-2 in the myocardium, as they pertain to two basic phenomena: ischemia-reperfusion injury and cardiac hypertrophy. The first category includes roles for FGF-2 in cardioprotection, the inflammatory response, angiogenesis and vascular remodeling, while the second includes myocyte hypertrophy, fibrosis, and gap junction functioning (conduction). Given the strong evidence for FGF-2 as both a cardioprotective and angiogenic agent, the therapeutic potential of FGF-2 in the ischemic myocardium is discussed.

Optical measurements of tissue oxygen saturation in lower limb wound healing.

This review has highlighted the role of oxygen in wound healing and in the mechanism of preventing infection. Optical measurements of tissue SO2 in wounds can provide valuable information, not only about the inflammatory state of the wound, but also about healing potential in ulcers and critical limb ischaemia. The technique is fast, non-invasive and can be used without the necessity for contact with the skin.

The effect of equine recombinant growth hormone on second intention wound healing in horses.

To evaluate the effect of intramuscular administration of recombinant equine growth hormone on healing of full thickness skin wounds on equine limbs. Experimental. Nine Standardbred horses. In study 1, standardized full thickness skin wounds (2.5 x 2.5 cm) were made over the dorsomedial aspect of the mid-cannon bone of 1 forelimb and 1 hindlimb in 9 horses. Wounds were bandaged without treatment (control subjects) and videorecorded twice weekly until healed. Then, in study 2, similar wounds were created on the opposite limbs; 6 horses were administered intramuscular recombinant equine growth hormone (10 microg/kg daily for 7 days, then 20 microg/kg daily for 49 days), and 3 horses (control subjects) were administered equivalent volumes of sterile water. Wounds were videorecorded twice weekly until healed. Wound healing variables were measured from the videorecordings using a computer software package and analyzed as a randomized complete block design factorial analysis of variance; significance was set at P <.05. No differences in the measured variables were detected between wounds in study 1 and the control wounds in study 2. In recombinant equine growth hormone-treated horses, wounds retracted more during treatment and contracted faster after treatment stopped when compared with wounds from untreated horses. No other treatment effects were detected. Recombinant equine growth hormone seemingly increases wound retraction. After treatment ceases, wound contraction increases. Intramuscular administration of recombinant equine growth hormone (10 microg/kg daily for 7 days, then 20 microg/kg daily for 49 days) does not appear to have any beneficial clinical effect on healing of equine limb wounds.

Effects of 25% propylene glycol hydrogel (Solugel) on second intention wound healing in horses.

To evaluate the effect of a commercially available 25% propylene glycol hydrogel preparation (Solugel; Johnson and Johnson Medical, North Ryde, Australia) on healing of full-thickness skin wounds on the distal aspect of the limb in horses. Experimental. Eight Standardbred horses. Standardized (2.5 x 2.5 cm) full-thickness skin wounds were created over the mid-dorsomedial aspect of both metacarpi in 8 horses. One wound in each horse was dressed with saline solution (0.9% NaCl) soaked gauze, and one was treated with Solugel under dry regular gauze; wounds were then bandaged with gauze-coated cotton wool and elastic adhesive bandages. Wounds were videorecorded and rebandaged twice weekly until healed. Wound healing variables were measured from the videorecordings using a computer software package and analyzed as a randomized complete block design with repeated measures. Where necessary variables were made positive for analysis; significance was set at P <.05. The area of the wound at the first bandage change did not vary between treated and untreated wounds. Treatment had no effect on the total rate of healing, rate of healing during the retraction phase of healing, rate of healing after the retraction phase was complete, or the amount the wounds retracted. Using this model of wound healing, Solugel had no effect on second intention healing of distal limb wounds in horses. Solugel does not appear to have any beneficial effect on healing of small full-thickness skin wounds to the distal limb of horses.

Unna and elastic postoperative dressings: Comparison of their effects on function of adults with amputation and vascular disease

Wong CK, Edelstein JE. Unna and elastic postoperative dressings: comparison of their effects on function of adults with amputation and vascular disease. Arch Phys Med Rehabil 2000;81:1191-8. Objectives: To compare the functional outcome associated with the use of Unna semirigid dressings (SRD) and elastic bandage soft dressings (ED) for adults with lower limb amputation. Design: Experimental design. Setting: Inpatient rehabilitation unit of an urban academic medical center. Participants: A successive series of adults with vascular disease who had lower limb amputation surgery. Subjects were randomly assigned to the SRD (12 patients with 12 recent amputations) or the ED (9 patients with 10 recent amputations) group. Subjects in each group were not significantly different except for age; those in the SRD group were somewhat older. Intervention: Subjects in the SRD group had Unna dressings applied to the amputation limb by physical therapists trained in the technique. Those in the ED group had elastic bandaging by therapists, nurses, family, and themselves, all of whom were trained in the technique. Results: Sixty-seven percent of the SRD group and 20% of those in the ED group were discharged from the rehabilitation unit ambulating with prostheses. Of those who received prostheses, time from admission to the rehabilitation unit to readiness for fitting averaged 20.8 days for the SRD group and 28.7 days for the ED group. Comparison of survival curves shows that the time from surgery to fitting in the SRD group was almost half that of the ED group; 30% of the SRD group was fitted within 34 days, whereas it took 64 days for the same percentage of the ED group to be fitted. Conclusions: Unna semirigid dressings are more effective in fostering amputation limb wound healing and preparing the amputation limb for prosthetic fitting. Subjects treated with SRDs were more likely to be fitted with prostheses and to return home walking with a prosthesis. © 2000 by the American Congress of Rehabilitation Medicine and the American Academy of Physical Medicine and Rehabilitation

The effectiveness of the haemodialysate Solcoseryl for second-intention wound healing in horses and ponies.

Second-intention healing of limb wounds in horses is often problematic. Solcoseryl is a protein-free, standardized dialysate/ultrafiltrate (HD) derived from calf blood, which has been shown to improve healing in both animals and humans. The efficacy of HD in the healing of deep wounds in horses and ponies was investigated. Deep wounds of 20 by 35 mm were created on both metatarsi (skin, subcutis, periosteum) and on both femoral biceps muscles (skin, subcutis, muscle) of five horses and five ponies. The wounds on one side were treated with HD, four times a week during the period that the wounds were bandaged and once daily thereafter. The wounds on the other side were left untreated. In the first 4 weeks of the healing period HD stimulated healing but inhibited healing thereafter. This pattern was significant for all wound groups (P < 0.001). Because of this change in effect, the overall effect on wound healing over the entire period was not significant (P = 0.77). HD stimulated healing initially by provoking a greater initial inflammatory response, faster contraction and faster formation of granulation tissue. Subsequently, HD inhibited healing because it significantly delayed epithelialization and caused protracted inflammation. The effects of HD were most pronounced in the horses. Because this study distinguished between contraction and epithelialization, it could be shown that HD stimulated contraction but inhibited epithelialization. Therefore, HD is useful in horses for the treatment of deep wounds during the initial phase of healing by second intention, i.e. during the first weeks when wound contraction can be expected. Treatment should be ceased when epithelialization becomes predominant.

Neuronal defects and delayed wound healing in mice lacking fibroblast growth factor 2.

Basic fibroblast growth factor (FGF2) is a wide-spectrum mitogenic, angiogenic, and neurotrophic factor that is expressed at low levels in many tissues and cell types and reaches high concentrations in brain and pituitary. FGF2 has been implicated in a multitude of physiological and pathological processes, including limb development, angiogenesis, wound healing, and tumor growth, but its physiological role is still unclear. To determine the function of FGF2 in vivo, we have generated FGF2 knockout mice, lacking all three FGF2 isoforms, by homologous recombination in embryonic stem cells. FGF2(-/-) mice are viable, fertile and phenotypically indistinguishable from FGF2(+/+) littermates by gross examination. However, abnormalities in the cytoarchitecture of the neocortex, most pronounced in the frontal motor-sensory area, can be detected by histological and immunohistochemical methods. A significant reduction in neuronal density is observed in most layers of the motor cortex in the FGF2(-/-) mice, with layer V being the most affected. Cell density is normal in other regions of the brain such as the striatum and the hippocampus. In addition, the healing of excisional skin wounds is delayed in mice lacking FGF2. These results indicate that FGF2, although not essential for embryonic development, plays a specific role in cortical neurogenesis and skin wound healing in mice, which, in spite of the apparent redundancy of FGF signaling, cannot be carried out by other FGF family members.

Macrophage recruitment during limb development and wound healing in the embryonic and foetal mouse.

Macrophages play a pivotal role in the adult inflammatory response to wounding. They are directly responsible for cellular debridement and, by providing a source of growth factors and cytokines, they recruit other inflammatory and fibroblastic cells and influence cell proliferation and tissue remodelling. In this paper we investigate the role of macrophages in clearing areas of programmed cell death in the developing embryo and also their role in embryonic and foetal wound healing. Immunocytochemistry using the monocyte/macrophage-specific monoclonal antibody, F4/80, reveals a close association between areas of programmed cell death in the remodelling interdigital regions of the mouse footplate and of F4/80-positive cells, suggesting that monocyte-derived macrophages, and not locally recruited fibroblastic cells, as previously believed, are responsible for phagocytosing and clearing areas of interdigital apoptosis. Our studies of wound healing reveal that macrophages are not recruited to, and therefore cannot be playing an active role in the healing of, excisional wounds made in the mouse embryo at any stage up until E14.5. Beyond this transition stage we see a significant recruitment of macrophages within 12 hours of wounding. We find that macrophages can be attracted to wounds in earlier embryos if the wound results in significant cell death such as after burning.

Second-Intention Healing

Second-intention healing (contraction and epithelization) is most appropriate for heavily traumatized, contaminated wounds that may have a prolonged debridement phase. Therapy during healing can promote rapid debridement and faster healing. Healing of lower limb wounds by second intention may be protracted, owing to poorer wound contraction and excessive granulation tissue production as compared with body wounds.

The Mesh Expansion Method of Suturing Wounds on the Legs of Horses

Wound closure utilizing tension‐relieving incisions located in the skin adjacent to the wound of horses was evaluated in three experiments. Healing of lower limb wounds was compared in four treatment groups; wounds sutured under tension, wounds sutured with 7 mm and 10 mm tension relieving incisions, and unsutured controls. The wounds sutured using 10 mm tension relieving incisions healed significantly faster than the other three groups (p &gt; 0.05). The mesh expansion technique was further evaluated in four‐week‐old granulating wounds. The technique was found to be unsuccessful because the tension relieving incisions that were used were too small and postoperative support was inadequate. The technique was evaluated in two clinical patients, both of which had large metatarsal lacerations. Tension relieving incisions of approximately 15 mm were made and full leg walking bar casts were applied. Both wounds had healed almost completely on removal of the casts four weeks postoperatively.