Effect of topical rh-TGF-beta 1 on second intention wound healing in horses

OBJECTIVE: To investigate the effects on wound healing of transforming growth factor-beta 1 as a topical treatment to full-thickness, excisional wounds of the distal limb of horses. DESIGN: A randomised block study using four horses, each with wounds assigned to four treatment groups. ANIMALS: Four adult Standardbred geldings. PROCEDURE: Four, 4 cm2, full-thickness wounds were created on the dorsomedial and dorsolateral aspect of the metacarpus or metatarsus of each limb of four horses, giving a total of 64 wounds. For each limb, wounds were randomly assigned to four treatment groups: no treatment (control), carrier (Methyl Cellulose gel), 50 ng/wound rhTGF-beta 1 in carrier, and 500 ng/wound rhTGF-beta 1 in carrier. Wounds were treated on day 0 and day 8. Effects of treatment were evaluated on the basis of the presence of exuberant granulation tissue requiring excision, number of times excision was required, total wound area, area of epithelialisation, area of granulation, and histological evaluation of biopsy samples of wounds on day 8 and excised wounds on day 21. RESULTS: Topical application of TGF-beta 1 at the two concentrations studied had no significant effect on the total area of wounds (P = 0.7), the area of granulation tissue (P = 0.78), the area of epithelialisation (P = 0.92), histological assessment or subjective clinical assessment of wounds. CONCLUSION: TGF-beta 1 had no beneficial effects on wound healing. Additional trials are needed to test if it has value for wound treatment in horses.     

Negative-Pressure Wound Therapy as Management of a Chronic Distal Limb Wound in the Horse

Wound healing in distal limb wounds with tissue loss in horses may be an expensive and long process due to prolonged inflammatory phase. Negative pressure wound therapy (NPWT) (also known as vacuum-assisted closure) is well established method of wound therapy in human plastic and reconstructive surgery for many years. It consists of a leak free bandage and a pump that applies subatmospheric pressure to the wound area. This report describes the successful use of the NPWT intended for the suppression of exuberant granulation tissue development of the wound on distal limb in a horse.     

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

OBJECTIVE: 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. STUDY DESIGN: Experimental. ANIMALS: Eight Standardbred horses. METHODS: 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. RESULTS: 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. CONCLUSIONS: Using this model of wound healing, Solugel had no effect on second intention healing of distal limb wounds in horses. CLINICAL RELEVANCE: Solugel does not appear to have any beneficial effect on healing of small full-thickness skin wounds to the distal limb of horses.     

The use of magnetic resonance imaging for the assessment of distal limb wounds in horses: A pilot study

The utility of magnetic resonance (MR) imaging in the evaluation of equine solar foot penetrations is well established. The objective of this pilot study was to evaluate the utility of MR imaging in assessment of equine distal limb wounds excluding solar penetrations. Low-field MR images of 23 horses that had previously sustained distal limb wounds were reviewed in consensus by two ECVDI diplomats. Structures (bone; synovial structure; subcutaneous tissue and skin; and ligament/tendon) were identified as normal or abnormal on MR images, radiographs and ultrasound images and reports. All abnormalities were described. The presence of artefacts and their effect on image interpretation were also noted for each modality. Comparisons were made between imaging modalities, and it was noted if MR imaging influenced case management. Abnormalities of the bone were identified in 26% of horses on MR images and 17% of horses on radiographs; there were no osseous abnormalities identified on radiographs that were not identified on MR images, and additional features and better characterisation of lesions were noted on MR images. Tendon/ligament abnormalities were identified in 57% horses on MR and 47% of horses on ultrasound images. Magnetic susceptibility artefacts compromised MR image interpretation in 17% of horses. MR imaging of equine distal limb wounds allowed identification of both osseous and tendon/ligament abnormalities in more cases than either radiography or ultrasonography, and altered case management in 20/23 horses. Although MR imaging should not replace conventional imaging, this study highlights that MR imaging of equine distal limb wounds can yield information not detected on conventional imaging which may direct treatment and affect prognostication.     

The effects of topical oxygen therapy on equine distal limb dermal wound healing

Topical oxygen therapy (TOT) has been used in human medicine to promote healing in chronic wounds. To test the efficacy and safety of TOT in horses, an experimental wound model was created by making 1 standardized dermal wound on each limb of 4 healthy horses (n = 16). Each wound was fitted with an oxygen delivery cannula and covered with a bandage. One limb of each front and hind pair was randomly assigned to the treatment group (fitted with an oxygen concentrator device), with the contralateral limb assigned to the control group (no device). Wound area, epithelial area, and contraction were measured every 3 to 4 d. Biopsy samples and culture swabs were taken on days 16 and 32 to evaluate angiogenesis, fibroplasia, epithelial hyperplasia, inflammation and bacterial growth. Mean healing time in treated wounds (45 d, range: 38 to 52 d) was not significantly different from that in the paired control wounds (50 d, range: 38 to 62 d). Topical oxygen therapy had little effect on dermal wound healing in this experimental wound model in healthy horses.     

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.     

Platelet-rich Plasma Combined With a Sterile 3D Polylactic Acid Scaffold for Postoperative Management of Complete Hoof Wall Resection for Keratoma in Four Horses

Keratoma is a nonmalignant horse tumor that grows in the space between the horn of the hoof and the distal phalanx. Keratoma causes lameness in the horse, and surgical excision is the treatment of choice. Four horses underwent removal of a keratoma by complete hoof wall resection. The remaining wound was treated with platelet-rich plasma (PRP) combined with a sterile three-dimensional polylactic acid scaffold. The PRP was applied at 3, 6, 9, 12, 15, and 18 days postoperatively. The surgical site was cleaned with gauzes and swabs soaked in Ringer’s lactate solution before applying PRP and the foot bandage. Healthy granulation tissue developed at 6–21 days postoperatively. The hoof wall defect was completely filled with new hoof wall within 6–8 months after surgery. All horses returned to their previous exercise level, and no recurrence of lameness was reported by the owner.     

Second-intention repair in the horse and pony and management of exuberant granulation tissue.

Second-intention repair is faster in ponies than in horses and faster in body wounds than in limb wounds. To a large extent, the differences between horses and ponies can be explained by differences in the local inflammatory response, which are a result of the functional capacity of leukocytes. In ponies, leukocytes produce more inflammatory mediators,resulting in better local defense, faster cellular debridement, and a faster transition to the repair phases, with more wound contraction. In horses,leukocytes produce fewer mediators, initiating a weak inflammatory response, which becomes chronic. This inhibits wound contraction and gives rise to the formation of exuberant granulation tissue. The anatomic environment that influences the inflammatory response and wound contraction most probably determines the differences between body and limb wounds. In body wounds, better perfusion results in faster initiation of the inflammatory phase. The weaker local resistance results in a greater degree of contraction. In limb wounds, particularly of horses, the initial inflammatory response is weak and wound contraction is restricted. Both factors give rise to chronic inflammation, which further inhibits wound contraction and promotes exuberant granulation tissue. The high incidence of exuberant granulation tissue in limb wounds of horses can thus be explained by the chronicity of the inflammatory response as well as by the common use of bandages during treatment. Chronic inflammation is often not recognized as a cause of exuberant granulation tissue. It must be prevented and treated to promote the healing process. Bandages and casts stimulate the formation of exuberant granulation tissue; however, they are advantageous in many respects and play an important role in support of the overall healing process.     

Computed tomography of nuchal ligament and semispinalis capitis tendon avulsions in a foal

Head trauma is a common injury in young horses. This case report describes a 10‐week‐old Thoroughbred foal with an open wound following trauma to the poll region, showing abnormal head carriage and depression. A computed tomography (CT) study was performed under general anaesthetic (GA). This identified a complete avulsion of the nuchal ligament (NL) and partial avulsions of the left and right semispinalis capitis (SS) tendons. Following diagnosis and conservative treatment the foal made a good recovery and 18 months later the filly had no detectable neurological or physical abnormalities. Computed tomography enabled an accurate and rapid diagnosis of a novel injury.     

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.     

Effect of autologous platelet-rich plasma application on cutaneous wound healing in dogs

This study was conducted to identify the effectiveness of platelet-rich plasma (PRP) and efficacy of intralesional injection as a method of application to acute cutaneous wounds in dogs. Healthy adult beagles (n = 3) were used in this study. Autologous PRP was separated from anticoagulant treated whole blood in three dogs. Cutaneous wounds were created and then treated by intralesional injection of PRP in the experimental group, while they were treated with saline in the control group on days 0, 2 and 4. The healing process was evaluated by gross examination throughout the experimental period and histologic examination on day 7, 14 and 21. In PRP treated wounds, the mean diameter was smaller and the wound closure rate was higher than in the control. Histological study revealed that PRP treated wounds showed more granulation formation and angiogenesis on day 7, and faster epithelialization, more granulation formation and collagen deposition were observed on day 14 than in control wounds. On day 21, collagen deposition and epithelialization were enhanced in PRP treated groups. Overall, PRP application showed beneficial effects in wound healing, and intralesional injection was useful for application of PRP and could be a good therapeutic option for wound management in dogs.     

Effects of mesenchymal stem cells isolated from amniotic fluid and platelet-rich plasma gel on severe decubitus ulcers in a septic neonatal foal

This paper documents the treatment of severe decubitus ulcers with amniotic fluid mesenchymal stem cells and platelets rich plasma (PRP) gel in a septic neonatal foal. The colt needed 25days of hospitalization: during this period ulcers were treated for 15days with mesenchymal stem cells (MSCs) plus PRP, PRP gel alone, or aloe gel. Healing was faster using MSCs+PRP, and at 7months an ulcer treated with aloe gel was still not completely healed.     

Effect of bandaging on second intention healing of wounds of the distal limb in horses

Objective   To evaluate the effect of a non-occlusive dressing incorporated in a 3-layer bandage on second intention healing of wounds of the distal portion of the limb.
Study Design   Untreated wounds in 33 adult horses used in four studies using the same wound-healing model conducted over 5 years.
Methods   Standardised, full-thickness wounds were made in the skin overlying the dorsomedial aspect of the mid-metacarpus; 17 horses were bandaged with a non-occlusive dressing covered by gauze-coated cotton wool that was compressed with adhesive tape; 16 horses were left unbandaged. Wounds were photographed weekly for 9 weeks and the images were analysed electronically.
Results   There were significant effects associated with bandage (P < 0.0001), week (P < 0.001), and bandage by week interaction (P < 0.0001). There was no difference in wound area at the first time-point after wound creation (P = 0.38). After week 1, there was a difference between bandaged and unbandaged wounds in wound area at each measurement until the end of the study. Bandaged wounds showed greater and more prolonged retraction. Unbandaged wounds retracted for 2 weeks before beginning to contract, whereas bandaged wounds continued to retract for 3 weeks. In bandaged wounds excess granulation tissue required regular trimming, but not in unbandaged wounds. There was no difference between groups in the total days to healing or the overall rate of healing.
Conclusions   These results should be treated with caution until validated with contemporaneous, controlled studies. Covering a wound with a non-occlusive dressing in a 3-layer bandage led to greater wound retraction, modulated the rate of wound contraction and promoted excessive granulation tissue. If excessive granulation tissue is excised regularly, bandaging has no effect on total time to healing.     

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

REASONS FOR PERFORMING STUDY: 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. HYPOTHESIS: The percentage take of Meek micrografts is higher than of other techniques; and rates of both wound contraction and epithelialisation are increased. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. POTENTIAL RELEVANCE: 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.     

Kinetics of healing of grafted and nongrafted wounds on the distal portion of the forelimbs of horses.

Full-thickness, circular, cutaneous wounds (5 cm in diameter) were created on the distal portion of the forelimbs of 6 horses. One wound on each horse was treated with 6 full-thickness punch grafts that were obtained from the horse's neck with a 6-mm skin biopsy punch and inserted in the graft sites on day 14 after wounding. The wound on the contralateral limb was not grafted. A combination of ticarcillin disodium and clavulanate potassium was applied to the wounds when bandages were changed to control bacterial infection. Areas of each wound were measured on days 1, 7, 9, 11, 13 through 15, 17 through 22, 24, 26, 29, and 32 after wounding. Three distinguishable phases of healing were observed (expansion, contraction, and epithelialization), and the time course of each phase was evaluated, using formulas of first-order processes. Rate constants of each phase were not significantly (P less than 0.05) affected by punch grafts.     

Laminin receptor 1 is differentially expressed in thoracic and limb wounds in the horse

Healing of wounds located on the distal limbs of horses is often complicated by retarded epithelialization and the development of exuberant granulation tissue (proud flesh). Treatments that definitively resolve this pathological process are still unavailable. Molecular studies of the repair mechanism might contribute to the development of new therapeutic strategies. The study presented herein aimed to clone the full length cDNA and to study the spatio-temporal expression profile of mRNA and protein for LAMR1, previously attributed a role in wound epithelialization, during the repair of body and limb wounds in the horse. Cloning was achieved by screening a cDNA library previously derived from 7-day wound biopsies. Expression was studied in unwounded skin and in samples from 1-, 2-, 3-, 4- and 6-week-old wounds of the body and limb. Temporal gene expression was determined by real time polymerase chain reaction (RT-PCR) while protein expression was mapped immunohistochemically. Full-length cDNA for equine LAMR1 was shown to be highly similar to that of other species. The mRNA expression of LAMR1 was significantly up-regulated only in thoracic wounds, 4 and 6 weeks following wounding (upon epithelialization). Cutaneous wounding induced protein expression at both locations. Our data suggest that up-regulation of LAMR1 protein might favour epithelialization during wound healing. However, its interaction with ligands other than laminin complicates data interpretation. Future studies should quantitatively verify the temporal expression of this protein in order to provide the basis for targeted therapies that might enhance epithelialization.     

Congenital bilateral aplasia of the metacarpophalangeal joints in a foal

A 2‐day‐old Warmblood filly was presented for examination of an angular limb deformity of the left front limb and an upright conformation of both metacarpophalangeal joints. Radiological examination revealed bilateral absence of the metacarpophalangeal joint space with fusion of the third metacarpal bone and first phalanx (synostosis). No treatment was undertaken. The filly was readmitted to the clinic 10 weeks later for bilateral front limb lameness. On radiological examination, the synostosis of the front metacarpophalangeal joints was still present. Physitis of the distal growth plate of the right third metacarpal bone and proximal growth plate of the right proximal phalanx, and an avulsion fracture of the palmaromedial and proximal aspect of the left middle phalanx, with a cystic like lesion on the medial aspect of distal first phalanx and proximal middle phalanx were diagnosed. Given the poor prognosis, the foal was subjected to euthanasia. Post mortem examination confirmed the absence of the metacarpophalangeal joint space with a trabecular bony union between the third metacarpal bone and the first phalanx. A rudimentary joint capsule was present at the level of the absent joints as well as a small zone of articular cartilage, which invaginated over a short distance into the dorsal trabecular bone on the right front limb. On the medial aspect of the left proximal interphalangeal joint, a focal defect of articular cartilage with exposure of subchondral bone was observed. This is the first case report of a foal born with congenital aplasia of both metacarpophalangeal joints. Congenital malformations should be considered as differential diagnosis in lame foals or foals born with angular or flexural limb deformities.     

Effect of a silicone-containing dressing on exuberant granulation tissue formation and wound repair in horses

OBJECTIVE: To determine the effect of a silicone dressing on the rate and quality of repair of limb wounds and compare microvascular occlusion and apoptosis in wounds treated with the silicone dressing and those treated with a conventional dressing in horses. ANIMALS: 5 horses. PROCEDURE: Horses received two 6.25-cm2 wounds on each metacarpus. Ten wounds were treated with a silicone dressing; the other 10 were treated with a control dressing. Quality of repair and wound size were evaluated at each bandage change. Time to healing and the number of excisions of exuberant granulation tissue were recorded. Biopsy specimens taken from healed wounds were evaluated semiquantitatively via histologic examination, p53 immunohistochemical analysis, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) to quantify apoptosis, and electron microscopic examination to measure microvessel luminal diameters. RESULTS: The silicone dressing surpassed the conventional dressing in preventing formation of exuberant granulation tissue and improving tissue quality. Microvessels were occluded significantly more often in wounds dressed with the silicone gel, which also diminished the expression of mutant p53, an indirect inhibitor of apoptosis, although greater apoptosis was not confirmed quantitatively by use of TUNEL. CONCLUSIONS AND CLINICAL RELEVANCE: Because the silicone dressing inhibited the formation of exuberant granulation tissue, it may be integrated in a management strategy designed to improve the repair of limb wounds in horses.     

Endothelial cell hypertrophy is associated with microvascular occlusion in horse wounds

Wound repair in horse limbs is often complicated by excessive fibroplasia and scarring. Occlusion of the microvessels populating the granulation tissue appears to be involved in the excessive accumulation of extracellular matrix during the repair of limb wounds. This study aimed to determine whether endothelial cell hypertrophy or hyperplasia, or both, contribute to microvascular occlusion and whether the pericyte is involved in this anomaly. We created 5 wounds, each 2.5 x 2.5 cm, on both forelimbs and on the body of 6 horses. One limb was bandaged to stimulate excessive wound fibroplasia. Weekly biopsy specimens were evaluated by transmission electron microscopy to measure microvessel luminal diameters and the surface area of endothelial cells and to count endothelial cells and pericytes. Microvessels were occluded significantly more often in limb wounds than in body wounds. The surface area of endothelial cells lining occluded microvessels (mean +/- standard error, 28.4013 +/- 1.5154 microm2) was significantly greater (P = 0.05) than that of cells lining patent microvessels (26.2220 +/- 1.5268 microm2). Conversely, neither the number of endothelial cells nor the number of pericytes differed between patent and occluded microvessels or between limb and body wounds. Furthermore, the wound location and the status of the microvessels (patent or occluded) did not alter the ratio of endothelial cells to pericytes. These data suggest that endothelial cell hypertrophy might play a role in the microvascular occlusion present in granulation tissue of limb wounds in horses, but the contribution of the pericyte remains obscure.