OB‐cadherin cloning and expression in a model of wound repair in horses

Reason for performing study: Horses suffer from a debilitating impediment in repairing wounds located on the lower limb that leads to the development of a fibroproliferative disorder (exuberant granulation tissue). This condition is a source of wastage since it often forces retirement from competition. Treatments that resolve or prevent this condition are still lacking, maybe due to deficient knowledge of the underlying molecular mechanisms. Fibroblast‐to‐myofibroblast conversion is an essential step allowing contraction during wound repair and is accompanied by an increase in OB‐cadherin expression. Objectives: To clone equine cadherin‐11 (CDH11) cDNA and to study its spatiotemporal expression profile during the repair of body and limb wounds, thereby contributing to a better understanding of the repair process. Methods: Cloning was by a PCR technique. Expression was studied in intact skin and in 1, 2, 3, 4 and 6‐week‐old wounds of the body and limb. Temporal CDH11 gene expression was determined by RT‐PCR while OB‐cadherin protein expression was mapped immunohistochemically. Results: Equine CDH11 is a highly conserved gene and protein. mRNA was not expressed in equine skin whereas the wound repair process was characterised by a significantly higher expression in the thorax than in limb samples. mRNA expression pattern was paralleled by protein data as confirmed by immunohistochemistry. Conclusions: The data suggest that deficient OB‐cadherin expression in the first phases of wound repair contributes to the excessive proliferative response seen in horse limb wounds. Potential relevance: Future studies should verify the quantitative, temporal expression of this protein in order to provide the basis for targeted therapies that might prevent the development of EGT in horse wound repair.     

Equine wound management: are there significant differences in healing at different sites on the body?

Abstract Trunk/body wounds heal rapidly with prominent Contraction while wounds on the limb commonly fail to heal or heal slowly by centripetal epithelialization. often with insignificant contraction. Chronic exuberant granulating wounds on the limbs heal well after grafting from donor sites on the trunk. Indolent wounds are less common but may granulate significantly following moist wound-healing management. Sarcoid transformation is an increasingly important cause of healing failure. Sarcoid transformations on the trunk are commonly verrucose while those on the limb are usually aggressive and fibroblastic. The primary objective of wound management should be to encourage rapid progression from acute inflammation to repair without intervention of chronic inflammation which is a significant factor in the pathophysiology of wound healing failure. Wounds fail to heal because there is disruption of the normal delicate balance of growth factors and inflammatory mediators. Wounds should be managed in such a way as to restore the balance of healing processes without damaging any of the cells involved in healing. Resumen Las heridas localizadas en el cuerpo o tronco curan rapidamente con contracción elevada, mientras que las de las extremidades no curan o curan lentamente por epitelización centrípeta, a menudo con contracción insignificante. Las heridas crónicas con tejido de granulación exhuberantes en las extremidades curan bien después de injertos a partir de áreas del tronco. Las heridas indolentes son menos frecuentes pero pueden granular significativamente después de un manejo húmedo de la curacion de la herida. La transformación sarcoidal es una causa cada vez más frecuente de fracaso de la curación. Las transformaciones sarcoidales en el tronco son generalmente de tipo verrucoso mientras que las de las extremidades son normalmente agresivas y fibroblásticas. El objetivo principal del manejo de heridas tendría que ser la estimulación de una progresión rápida de una inflamación aguda a la resolución sin intervención de inflamación crónica, que es un factor significativo en la patofisiología del fracaso en la curación de heridas. Las heridas no curan porque se produce una alteración en el frægil equilibrio normal de factores de crecimiento y mediadores inflamatorios. Las heridas tendrian que ser manejadas de forma que se restaure el equilibrio del proceso curativo sin dañar ninguna de las células implicadas en la curacion. [Knottenbelt, D.C. Equine wound management: are there significant differences in healing at different sites on the body? (Manejo de heridas equinas: existen diferencias significativas en la curación en diferentes areas cutaneas?). Veterinar.): Dermatology 1997; 8 : 273–290] Zusammenfassung Wunden am Rumpf heilen schnell und mit deutlicher Kontraktion, während sich Wunden an den Gliedmassen oft nur geringfügig zusammenziehen und entweder gar nicht oder nur langsam vom Rande her epithelisieren. Chronische, stark wuchernde Wunden an den Gliedmassen heilen nach Gewebetransplantation vom Rumpf zufriedenstellend. Indolente Wunden sind weniger häfig, können aber nach benetzender Wundversorgung deutlich granulieren. Sarkoide Transformation ist ein wichtiger Grund für Wundheilungstörungen. Sarkoide Transformationen am Rumpf sind gewöhnlich warzig, jene an den Gliedmassen üblicherweise aggressiv und fibroblastisch. Das wichtigste Ziel der Wundversorgung sollte es sein, eine schnelle Entwicklung von akuter Entzündung zur Heilung zu fördern, ohne in die chronische Entzündung einzugreifen, die ein massgeblicher Faktor in der Störung der Windheilung darstellt. Der Grund für eine nichtheilende Wunde ist eine Störung des enipfindlichen Gleichgewichts zwischen Wachstumsfaktoren und Entzündungsmediatoren. Wundversorgung soll das Gleichgewicht des Heilungsprozesses wiederherstellen, ohne Zellen zu beschädigen, die zur Wundheilung beitragen. [Knottenbelt, D.C. Equine wound management: are there significant differences in healing at different sites on the body? (Wundversorgung beim Pferd: Sind signifikante Heilungsunterschiede an verschiedenen Körperstellen vorhanden?). Veterinary Dermatology 1997; 8 : 273–290] Résumé Les plaies affectant le tronc cicatrisent rapidement avec une contraction proéminente tandis que les plaies des membres cicatrisent ma1 ou lentement avec une épithélialisation centripète, et souvent avec une contraction insignifiante. Les plaies présentant une granulation chronique exhubérante sur les membres cicatrisent bien après greffe à partir d'un fragment prélevé sur le tronc. Les plaies atones sonl moins fréquentes, mais peuvent présenter un tissu de granulation significatif après application de pansements humides. La transformation en sarcoïde est une cause de plus en plus importante d'échec de cicatrisation. Les transformations en sarcoïdes sur le tronc sont fréquemment verruqueuses, alors que celles localisées sur les membres sont généralement agressives et fibroblastiques. Le but premier du traitement d'une plaie est de promouvoir la progression rapide d'une inflammation aigüe en cicatrisation sans passage par l'inflammation chronique qui est un facteur significatif dans la pathophysiologie de l'échec de cicatrisation des plaies. Les plaies ne cicatrisent pas parce qu'il y a un déséquilibre de la balance délicate entre facteurs de croissance et médiateurs de l'inflammation. Les plaies doivent être traitées de manière à restaurer l'équilibre des processus de cicatrisation sans altération des cellules impliquées dans l'inflammation. [Knottenbelt, D.C. Equine wound management: are there significant differences in healing at different sites on the body? (Traitement des plaies équines: y-a-t-il des différences significatives de cicatrisation en fonction de la topographie corporelle?). Veterinary Dermatology 1997; 8 : 273–290]     

A novel mouse wound model for scar tissue formation in abdominal muscle wall

Hypertrophic scars found on the human body rarely develop in experimental animals, possibly due to their looser skin structure. This makes it difficult to understand the genesis of scar lesions. Therefore, appropriate animal models are urgently needed. In this study, we established a novel experimental model of a scar-forming wound by resecting a small portion of the abdominal muscle wall on the lower center of the abdomen in C57BL/6N mice, which are exposed to contractive forces by the surrounding muscle tissue. As a low-tension control, a back skin excision model was used with a splint fixed onto the excised skin edge, and granulation tissue formed on the muscle fascia supported by the back skeleton. One week after the resection, initial healing reactions, such as fibroblast proliferation, occurred in both models. However, after 21 days, lesions with collagen-rich granulation tissues, which were also accompanied by multiple nodular/spherical-like structures, developed only in the abdominal wall model. These lesions were analogous to scar lesions in humans. Therefore, the animal model developed in this study is unique in that fibrous scar tissues form under physiological conditions without using any artificial factors and is valuable for studying the pathogenesis and preclinical treatment of scar lesions.     

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.     

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.     

Use of negative pressure wound therapy in three horses with open,infected olecranon bursitis

This article describes the clinical use of negative pressure wound therapy (NPWT) in three horses with an open and infected subcutaneous olecranon bursa, in which prior surgical excision followed by primary closure was unsuccessful. The wounds filled in rapidly with granulation tissue and contracted during therapy. All wounds subsequently went on to heal without complications. The use of NPWT was well tolerated in all three horses and facilitated healing in a difficult area.     

Cutaneous wound healing in the cat: a macroscopic description and comparison with cutaneous wound healing in the dog

OBJECTIVE: To describe the macroscopic features of first and second intention cutaneous wound healing in the cat and compare with the dog. STUDY DESIGN: Experimental study. ANIMALS: Domestic shorthaired cats (6) and beagle dogs (6). METHODS: Square, open cutaneous wounds created on the dorsal aspect of the thorax were evaluated for 21 days for temporal and spatial development of granulation tissue, wound contraction, epithelialization, and total healing. To evaluate first intention healing, breaking strength of sutured linear cutaneous wounds was measured at 7 days post-wounding. Laser-Doppler perfusion imaging was used to measure cutaneous perfusion. RESULTS: First intention healing: sutured wounds in cats were only half as strong as those in dogs at day 7 (0.406 versus 0.818 kg breaking strength). Second intention healing: cats produced significantly less granulation tissue than dogs, with a peripheral, rather than central distribution. Wound epithelialization and total wound healing (total reduction in open wound area from contraction and epithelialization) were greater for dogs than for cats over 21 days. Wound contraction on day 7 was greater for dogs, but not on day 14 or 21. Cutaneous perfusion was initially greater for dogs than for cats, but no differences were detected after day 7. CONCLUSIONS: Significant, previously unreported differences in cutaneous wound healing exist between cats and dogs. In general, cutaneous wounds in cats are slower to heal. Cats and dogs also appear to use different mechanisms of second intention healing. In cats wounds close mainly by contraction of the wound edges, whereas in dogs wounds close more from central pull, and epithelialization. CLINICAL RELEVANCE: Surgeons should view the cat as a unique species, which presents its own special challenges in wound healing, and should take this into account when planning treatment of feline wounds, either by primary closure, or by second intention healing.     

The mast cell in wound healing

This review describes the role of the mast cell in the pathobiology of skin healing. After illustrating its main morphofunctional characteristics, with special reference to the dog and cat, we consider the involvement of the mast cell in the various phases of skin repair. With the aid of a wide array of newly formed or preformed mediators released by degranulation, the activated mast cell controls the key events of the healing phases: triggering and modulation of the inflammatory stage, proliferation of connective cellular elements and final remodelling of the newly formed connective tissue matrix. The importance of the mast cell in regulating healing processes is also demonstrated by the fact that a surplus or deficit of degranulated biological mediators causes impaired repair, with the formation of exuberant granulation tissue (e.g. keloids and hypertrophic scars), delayed closure (dehiscence) and chronicity of the inflammatory stage.     

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.     

Results and complications of a novel technique for primary castration with an inguinal approach in horses

Reasons for performing study: Complications associated with equine castration can have medical and financial consequences. This retrospective study investigated a novel method of castration via an inguinal approach in mature stallions and compared the incidence of complications with other methods. Hypothesis: Castration via an inguinal approach has a low complication rate at the site of surgery compared with other castration techniques. Methods: Mature stallions (n = 238) were castrated under general anaesthesia in dorsal recumbency using an inguinal approach. The vaginal process was incised, the spermatic cord ligated twice and the testis removed. After suturing, the vaginal process and one or 2 layers of fascia, the subcutis and cutis were closed in a simple continuous pattern. Results: Five of 238 (2.1%) horses had post operative haemorrhage and a haematoma in the scrotal region, which required additional treatment. All horses made a full recovery. Five of 238 (2.1%) of the horses had a post operative respiratory infection, which resolved with antibiotic therapy. Sixteen of 238 (8.8%) had transient signs of colic shortly after surgery. Conclusion: This technique of castration with an inguinal approach had a low incidence of complications at the site of surgery compared with other methods. An inguinal approach and leaving the vaginal tunic in situ may cause less soft tissue trauma than a scrotal approach.     

Meet the corneal myofibroblast: the role of myofibroblast transformation in corneal wound healing and pathology

In recent years the role of the myofibroblast in corneal wound healing has been extensively studied. The transformation of the corneal keratocyte to the fibroblast and myofibroblast phenotypes is a critical process in normal and pathologic corneal wound healing. This review will introduce this important transformation and its role in corneal wound healing and pathology.     

Models in vivo of wound healing in the horse and the role of growth factors

Abstract Wound models attempt to simulate the natural healing processes in wounds. However, all models have significant limitations due to the complexity of the tissue repair process. Much can be learned from wound models in vitro by the use of cell culture techniques. The horse can provide a suitable naturally occurring model of chronic wound healing because it has many similarities to wound healing encountered in human medicine. The tissue architecture was investigated with regard to extracellular matrix and growth factor distribution during wound healing and growth factors were consistently present in the wound area. Biochemical investigations revealed increased levels of hydroxyproline, collagen, and TGFβ1 in exuberant granulation tissue. Equine wound models were established in vitro using cell culture techniques and growth factors had significant effects on the growth of the cells and their ability to synthesize collagen. Two gelatinases (MMP-2 and MMP-9) were detected in the tissues and wound fluid samples investigated. Zusammenfassung Wundmodelle haben zum Ziel, die natürlichen Heilungsprozesse in Wunden zu simulieren. Allen Modellen sind durch den komplexen Gewebsheilungsprozess deutliche Grenzen gesetzt. Durch die Verwendung von Zellkulturtechniken kann von Wundmodellen in vitro viel abgeleitet werden. Das Pferd stellt wegen der vielen Ähnlichkeiten zur humanmedizinischen Wundheilung ein geeignetes Modell für chronische Wundheilung dar. Die Gewebsarchitektur wurde bezüglich der Verteilung von extrazellulärer Matrix und von Wachtumsfaktoren während der Wundheilung untersucht; Wachtsumsfaktoren waren ständig in der Wunde vorhanden. Biochemische Untersuchungen ergaben erhöhte Hydroxyprolin-, Kollagenund TGFβ1-Spiegel. Wundmodelle beim Pferd in vitro und Zellkulturtechniken wurden entwickelt; Wachstumsfaktoren hatten deutliche Wirkung auf die Zellen und ihre Fahigkeit zur Kollagensynthese. Zwei Gelatinasen (MMP-2 und MMP-9) wurden im Gewebe identifiziert und Wundflüssigkeitsproben untersucht. [Cochrane, C.A. Models in vivo of wound healing in the horse and the role of growth factors. (Wundheilungsmodelle in vivo beim Pferd und die Rolle von Wachstunisfaktoren). Veterinary Dermatology 1997; 8 : 259–272] Resumen Los modelos de heridas intentan estimular el proceso natural de curación de heridas. Sin embargo, todos 10s modelos presentan limitaciones considerables debido a la complejidad del proceso de curación de heridas. Se puede aprender mucho de modelos de heridas in vitro mediante el uso de técnicas de cultivo celular. El caballo puede suponer un modelo natural adecuado de curación crónica de heridas ya que se asemeja a la curación de heridas en medicina humana. Se investigó la arquitectura tisular en referencia a la matriz extracelular y la distribución de factores de crecimento viendo que los factores de crecimiento se encontraban presentes de forma constante en el área de la herida. Las investigaciones bioquímicas revelaron incremento en los niveles de hidroxiprolina, colágeno y TGFbl en tejido de granulación exhuberante. Se establecieron modelos in vitro de heridas equinas utilizando técnicas de cultivo celular y los factores de crecimiento tuvieron efectos significativos en el crecimiento de las células y su capacidad de sintetizar colágeno. Se detectaron dos gelatinasas (MMP-2 y MMP-9) en 10s tejidos y muestras de fluidos de las heridas investigadas. [Cochrane, C.A. Models in vivo of wound healing in the horse and the role of growth factors. (Modelos de curacion de heridas in vivo en el caballo y el papel de 10s factores de crecimiento). Veterinary Dermatology 1997; 8 : 259–272] Résumé Les modèles de plaies tentent de simuler les processus naturels de cicatrisation. Cependant tous les modèles ont des limitations significatives dues à la complexité des processus de réparation des tissus. Beaucoup peut être étudié sur des modèles de plaies in vitro suite à l'utilisation des techniques de culture cellulaire. Le cheval peut constituer un modèle nature1 fiable de cicatrisation chronique de plaie de par les nombreuses similarités qu'il partage avec la cicatrisation des plaies en médecine humaine. L'architecture tissulaire a étéétudiée concernant la matrice extracellulaire et la distribution des facteurs de croissance pendant la cicatrisation, et les facteurs de croissance se sont montrés présents de façon consistante dans la zone de la plaie. Des investigations biochimiques ont révélé une élévation des taux d'hydroxyproline, collagène, et TGFβ1 dans la granulation tissulaire exubérante. Des modèles équins de plaies ont étéétablis in vivo en utilisant des techniques de culture cellulaire et les facteurs de croissance ont montré des effets significatifs sur la croissance des cellules et leur capacité de synthétiser le collagène. Deux gélatinases (MMP-2 et MMP-9) ont été détectées dans les tissus et les échantillons de fluide des plaies ont été analysés. [Cochrane, C.A. Models in vivo of wound healing in the horse and the role of growth factors. (Modeles in vivo de cicatrisation des plaies chez le cheval et role des facteurs de croissance). Veterinary Dermatology 1997; 8 : 259–272]     

The use of intraluminal stents for tracheal collapse in two horses: Case management and long‐term treatment

Tracheal collapse is an uncommon clinical disorder in horses but when present can be difficult to correct. Various medical and surgical procedures to correct tracheal collapse have been described in horses with variable success. Recently, the use of an intraluminal stent has been described as a treatment for tracheal collapse in a miniature horse. The long‐term management, utilising intraluminal stents, in 2 miniature horses with tracheal collapse is presented here. In particular, various complications as a result of intraluminal stent placement are described, the most persistent being the formation of granulation tissue at various regions of the stents. Multiple methods of combating granulation tissue in this situation also are discussed.