Effect of wound location and the use of topical collagen gel on exuberant granulation tissue formation and wound healing in the horse and pony

Preformed collagen gel was topically applied to cutaneous wounds of the equine dorsal fetlock (thoracic limb) and metatarsal regions to evaluate the effect on exuberant granulation tissue production and wound healing. In 6 horses and 3 ponies (less than 140 cm high at the withers and less than 365 kg), 36 standardized cutaneous limb wounds were surgically induced (4 wounds/animal); 18 wounds were treated topically with collagen gel, and 18 wounds were not treated (controls). Collagen gel was initially applied to the wound at 0, 2, or 7 days after wound formation (groups 1, 2, and 3, respectively). Four measurements were regularly made: amount of wound contraction and the size of the granulation bed, epithelial covering, and total wound. Sequential skin and wound biopsies were evaluated histologically to assess wound healing. Using a computer, data were analyzed for differences in the 4 measurements between treated and control wounds, between fetlock wounds and metatarsal wounds, and among groups 1, 2, and 3. Analyses were performed on days 15 and 45 of wound healing and on the final day of healing. A significant difference (P greater than 0.05) in the production of exuberant granulation tissue, rate of epithelialization, or degree of wound contraction was not detected between the collagen-treated and control wounds. Total healing time and final scar size were similar. Wound healing patterns were significantly different (P less than 0.05) in the fetlock wounds and metatarsal wounds. All wounds enlarged up to day 15 with fetlock wounds enlarging significantly more than did the metatarsal wounds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Management of exuberant granulation tissue

Exuberant granulation tissue is common in large, lower limb wounds of horses, particularly horses of large body size. Methods of control include chemical cautery, cryogenic surgery, and surgical resection. Surgical resection is preferred because it is easy to perform, provides tissue for histologic evaluation, and preserves the epithelial margin. Effective treatments to prevent the formation of granulation tissue include leaving granulating wounds open to the air or, possibly, bandaging with topical steroids. Bandaging or casting may promote exuberant granulation tissue in wounds in which it has already formed, but bandaging and casting are still important in the early management of lower limb or hock wounds in horses. The use of skin grafts or delayed secondary wound closure is frequently necessary to prevent the recurrence of exuberant granulation tissue.     

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.     

Use of a neodymium:yttrium-aluminum-garnet laser to remove exuberant granulation tissue from the esophagus of a horse

Use of a neodymium:yttrium-aluminum-garnet laser to remove exuberant granulation tissue from the esophagus of a horse A 4-year-old Quarter Horse mare was evaluated because of recurrent esophageal obstruction. Endoscopic examination revealed a proliferative mass in the esophagus approximately 55 cm aborad to the nares. Histologic examination of biopsy specimens revealed diffuse suppurative esophagitis and granulation tissue with no evidence of neoplasia. The mass was excisedand ablated transendoscopically with a neodymium:yttrium-aluminum-garnet laser. During a follow-up examination 2 weeks after the final laser procedure, the mass was no longer apparent, and the esophagus appeared healed. Six months after the final examination, there had been no recurrence of the mass. In this horse, transendoscopic laser excision was efficacious and avoided problems associated with an esophagotomy.     

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.     

The use of acupuncture beads to control exuberant granulation tissue in equine skin wounds: A preliminary study

Skin lacerations on the hind legs of horses often cause large defects on the cranial surface of the hock and cannon regions. The treatment of these present significant logistic problems due to the amount of movement of the area and the difficulties of bandaging and immobilisation, as well as a tendency to form exuberant granulation tissue (or ‘proud flesh’) Acupuncture beads were used as a means of suppressing granulation and maximising epithelial ingrowth, using the principle that ‘acupuncture has been shown to help bring the body back towards normality’. The aim in these cases was healing of the skin lesions with the least effort and cost possible. The technique eliminated the need for resection of any granulation tissue after the beads were implanted, the amount of bandaging was drastically reduced and eliminated in a lot of cases, and the results were cosmetically very acceptable and functional. Digital photography and measurements of the changes in the size of the lesions were used to document the results. This study shows that the formation of exuberant granulation tissue or ‘proud flesh’ in equine skin wounds on the cranial surface of the hock and cannon bone can be controlled by a single acupuncture bead treatment. Long‐term follow‐up of the cases over 18+ months confirms the ongoing effects of the acupuncture beads, resulting in the completion of wound healing and reduction of fibrosis.     

Effects of topical application of antimicrobials and bandaging on healing and granulation tissue formation in wounds of the distal aspect of the limbs in horses

OBJECTIVE: To determine whether povidone iodine ointment or 2 forms of silver sulfadiazine applied topically to wounds of the distal aspect of the limbs in horses affect the rate of second intention healing and to evaluate the additional influence of bandaging with these antimicrobials on granulation tissue formation. ANIMALS: 6 healthy adult horses. PROCEDURE: Six standardized 2.5-cm2 skin wounds/horse were distributed between the dorsomedial surfaces of the metacarpi and metatarsi. One of the following 6 treatments was applied to each wound: 1% silver sulfadiazine cream with bandage, 1% silver sulfadiazine slow-release matrix with bandage, 1% silver sulfadiazine slow-release matrix without bandage, povidone-iodine ointment with bandage, untreated control with bandage, and untreated control without bandage. Wound area, granulation tissue area, and perimeter were measured by use of planimetry software applied to digital images. Exuberant granulation tissue was excised when present. Days until healing, rate of healing parameter, rate of contraction, and epithelialization were compared among wound treatment groups. RESULTS: Healing parameters and mean days to healing did not differ significantly among any of the wound treatment groups. Percentage wound contraction and rate of epithelialization were similar among wound treatments. All bandaged wounds produced exuberant granulation tissue, which was surgically excised; none of the unbandaged wounds produced exuberant granulation tissue. CONCLUSIONS AND CLINICAL RELEVANCE: When exuberant granulation tissue is removed, rates of epithelialization and wound contraction were not different among wound treatment groups, whether bandaged or unbandaged. Topical application of 1% silver sulfadiazine slow-release matrix on unbandaged wounds induced the same result as medications applied beneath bandages, but without exuberant granulation tissue formation.     

Use of cancellous bone grafting to promote granulation tissue in a distal limb wound in a dog

A 5‐year‐old female spayed Shetland sheepdog presented for evaluation of bandage sores on the left pelvic limb after a splint was placed to stabilise a digit fracture incurred approximately 2 weeks previously. Multiple areas of necrosis were identified upon bandage removal. After debridement of devitalised tissue, a 4 cm wound on the plantar surface of the metatarsal bones (with exposed bone and tendons) remained. The Papineau technique (which involves removal of infected or necrotic tissue, placement of autogenous cancellous bone graft within a wound, and delayed skin closure) was used to treat this wound. Use of bone graft in this manner aims to promote early granulation tissue formation and reduce the time until wound reconstruction can be performed. Autogenous cancellous bone graft was harvested from the proximal humerus and placed within the metatarsal wound. Once a healthy granulation tissue bed was noted to cover the bone graft, a meshed full thickness skin graft was placed. To our knowledge, this is the first report of the Papineau technique being used for this purpose in a dog.     

Tunnel grafting for wound repair in horses: a novel technique in graft production and placement

There are several skin grafting methods described in the human and animal literature. Currently, there are five types of free grafts used in horses: pinch and punch grafts, split and full-thickness sheet or mesh grafts and tunnel grafts. Published methods of tunnel grafting describe the use of alligator forceps. The alligator forceps create a poor tunnel and are excessively traumatic to the granulation bed. This technique utilised a 13G Jamshidi needle that was placed across the granulation bed and created a uniform tunnel. The Jamshidi needle was atraumatic to the granulation bed increasing the opportunity for graft survival. A twin bladed scalpel allowed for the quick creation of uniform width grafts. Removal of the overlying tunnel ‘roof’ took place 5–14 days later to allow graft expansion. This case series included five horses with distal limb wounds and one with a wither injury. Four horses required general anaesthesia for graft placement and three required general anaesthesia for the removal of the tunnel roof. The acceptance of the grafts varied from 70% to 100%. Graft expansion to cover the granulation tissue took 2–5 months. This case series demonstrates that this technique of graft production and placement is an easy method for achieving successful skin grafting. Compared to other graft types, tunnel grafts are more readily accepted. Cosmetic and functional results achieved are better than those with pinch and punch grafts. Tunnel grafting does not require expensive equipment or advanced training, and in some cases can be performed under standing sedation.     

Influence of wound shape on wound contraction in horses.

Three sets of paired circular and square full-thickness skin wounds were made on the dorsum of the metacarpus (n = 48) of 8 horses. Each wound was 6.25 cm2 in area. The wounds were treated topically with an ointment, nonadherent dressing, and bandaged with a snug elastic wrap. Wounds were photographed every other day until healing was complete. Wound areas were measured and exponential and linear wound healing models were applied to the wound healing data generated. Wound healing variables measured for each wound were: number of days to healing, maximal size attained, rate of wound contraction (calculated by use of first-order and linear models), final wound size, and percentage of wound that healed by contraction. The exponential model fit the data significantly better than the linear model. The maximal size attained by circular wounds was significantly smaller than the maximal size attained by square wounds. Wound shape did not influence the rate of wound healing. On the basis of our findings, conversion of circular defects to square defects would not speed wound healing.     

Preliminary observations on expression of transforming growth factors beta1 and beta3 in equine full-thickness skin wounds healing normally or with exuberant granulation tissue

OBJECTIVE: To determine whether transforming growth factor (TGF)-beta1 and -beta3 expression differs between equine limb wounds healing normally and those healing with experimentally induced exuberant granulation tissue (EGT). STUDY DESIGN: Six wounds were created on the lateral aspect of both metacarpi of each horse; one forelimb was untreated, and the other was bandaged to stimulate the development of EGT. Sequential wound biopsies allowed comparison of growth factor expression between the two types of wound. ANIMALS: Four horses (2 to 4 years of age; 350 to 420 kg). METHODS: Wounds were assessed grossly, histologically, and by enzyme-linked immunosorbent assay (ELISA) for TGF-beta1 and -beta3 expression at 12 and 24 hours and 2, 5, 10, and 14 days postoperatively. RESULTS: Bandaged wounds developed EGT. In all wounds, TGF-beta1 peaked early and remained elevated at 14 days. Peak TGF-beta1 concentration was higher in wounds with EGT, but not significantly so. Expression of TGF-beta3 differed from TGF-beta1, with peak TGF-beta3 concentrations being delayed. Concentrations of TGF-beta3 were higher in wounds healing normally, but this difference was not significant. CONCLUSIONS: During both normal and exuberant wound repair, the expression of TGF-beta1 occurred earlier than TGF-beta3 expression. Wounds healing with EGT tended to have higher concentrations of fibrogenic TGF-beta1 and lower concentrations of antifibrotic TGF-beta3 than wounds healing normally, although these differences were not statistically significant. CLINICAL RELEVANCE: This study suggests that the production of EGT in bandaged wounds may be related to increased expression of fibrogenic TGF-beta1 and decreased expression of antifibrotic TGF-beta3. Further investigation of the roles of TGF-beta1 and -beta3 may be important in understanding the molecular control of EGT in horses.     

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.     

Proteoglycan synthesis and content in articular cartilage and cartilage repair tissue in horses

Hexosamine concentration, DNA concentration, and [35S]sulfate incorporation for articular cartilage obtained from various sites in the metacarpophalangeal and carpal joints of horses were measured. The same measurements were made on the repair tissue filling full-thickness articular defects in the intermediate carpal bone and on cartilage surrounding partial-thickness defects 6 weeks after the defects were created arthroscopically. Cellularity (measured as DNA concentration), proteoglycan content (measured as hexosamine concentration), and proteoglycan synthesis (measured as [35S]sulfate incorporation) varied according to the site sampled. Cartilage from the transverse ridge of the head of the third metacarpal bone and the radial facet of the third carpal bone had the lowest hexosamine concentration, whereas rate of proteoglycan synthesis was lowest in cartilage from the transverse ridge of the head of the third metacarpal bone and the distal articular surface of the radial carpal bone. Repair tissue filling a full-thickness cartilage defect at 6 weeks was highly cellular. It was low in proteoglycan content, but was actively synthesizing these macromolecules. In contrast, the cartilage surrounding a partial-thickness defect was unchanged 6 weeks after the original defect was made.     

Practical treatment of body and open leg wounds of horses with bovine collagen,biosynthetic wound dressing and cyanoacrylate

Horses with severe and deep lacerations are represented by ten cases in which treatment emphasized the use of bovine collagen preparations to promote controlled second-degree repair. Traumatized areas were tarsal, metatarsal, neck, forearm, metacarpal and pastern. Wound size changes were recorded. Depending on the wound type, the site was treated with antibiotic-steroid ointment, organic acid cream, sterile collagen particles, suspension or dressings protected by hydrogel dressing, non-adherent pads, occlusive skin dressings, roll gauze and elastic tape. In three cases, a fiberglas cast was applied over a hind leg wound and lacerated tendon for stability. When controlled granulation of the deeper wounds reached skin level, the area often was stabilized by only cyanoacrylate spray. As these cases presented a wide range of trauma each with a unique history, healing rates were based on initial measurements. An overall progression of wound reduction occurred at a predictable rate. The exogenous collagen formulations were used to stimulate controlled granulation, ie. to “jump start” the healing process. Collagen particles, suspension or dressings were packed into depressions, placed under suture lines, secured over abraded tissue, and placed under protective bandage or cast. To further evaluate the use of cyanoacrylate tissue spray in wound treatments, an additional ten cases are presented. The variety of wounds were produced experimentally in Center-owned ponies or provided as clinical cases. Wound size changes and healing progress were recorded. Wounds occurred on the neck, abdomen, metacarpal, metatarsal, fetlock and pastern areas. Depending on wound type, the site was treated with cyanoacrylate only; or treated as above until controlled granulation attained skin level. In one case punch grafts of skin were transferred from one foreleg to the opposite with the horse standing. Cyanoacrylate spray provided a water proof barrier protecting the wound from dirt, debris and insects as well as stabilizing full-thickness skin lacerations by bridging normal to traumatized skin allowing uninterrupted granulation and epithelialization. The use of a neck cradle prevented wound disturbance and stall confinement aided stabilization.     

Effects of a proprietary topical medication on wound healing and collagen deposition in horses

Full-thickness skin wounds were created on the dorsum of both metacarpi in 8 horses. Three topical treatment regimens were studied. All wounds were bandaged with a nonadherent dressing, which was held in place with a snug elastic wrap. Group-A wounds were treated with a proprietary topical wound medication that consisted of a spray and an ointment. Group-B wounds were treated with the same regimen, except the putative active ingredients in the ointment were omitted. Group-C wounds were treated with a dry nonadherent bandage only. Wound dressings were changed every day and the limbs were photographed every other day until the wounds were healed. Specimens of normal skin and biopsy specimens of healed wounds were examined histologically and were assayed for hydroxyproline content. Wound healing measurements quantitated for each wound were number of days to healing, maximal wound size attained, day wound contraction commenced, day epithelium first noticed, rate of wound contraction, final wound size, and fraction of the wound that healed by contraction. The cosmetic appearance of the healed wounds was also graded. Significant differences were not noticed in hydroxyproline content, histologic appearance, or any of the wound healing measurements between treatment groups. The cosmetic appearance of healed group-A and -B wounds was significantly better than the appearance of group-C wounds. The topical treatment regimens studied neither enhanced nor inhibited wound healing in this study.     

Comparison of a new laser beam wound camera and a digital photoplanimetry-based method for wound measurement in horses

The aim of this study was to compare the accuracy, precision, inter- and intra-operator reliability of a new laser beam (LB) wound camera and a digital photoplanimetry-based (DPB) method for measuring the dimensions of equine wounds. Forty-one wounds were created on equine cadavers. The area, circumference, maximum depth and volume of each wound were measured four times with both techniques by two operators. A silicone cast was made of each wound and served as the reference standard to measure the wound dimensions.The DPB method had a higher accuracy and precision in determining the wound volume compared with the LB camera, which had a higher accuracy in determining the wound area and maximum depth and better precision in determining the area and circumference. The LB camera also had a significantly higher overall inter-operator reliability for measuring the wound area, circumference and volume. In contrast, the DPB method had poor intra-operator reliability for the wound circumference. The LB camera was more user-friendly than the DPB method.The LB wound camera is recommended as the better objective method to assess the dimensions of wounds in horses, despite its poorer performance for the measurement of wound volume. However, if the wound measurements are performed by one operator on cadavers or animals under general anaesthesia, the DPB method is a less expensive and valid alternative.     

Effect of adipose-derived nucleated cell fractions on tendon repair in horses with collagenase-induced tendinitis

OBJECTIVE: To assess the potential of adipose-derived nucleated cell (ADNC) fractions to improve tendon repair in horses with collagenase-induced tendinitis. ANIMALS: 8 horses. PROCEDURES: Collagenase was used to induce tendinitis in the superficial digital flexor tendon of 1 forelimb in each horse. Four horses were treated by injection of autogenous ADNC fractions, and 4 control horses were injected with PBS solution. Healing was compared by weekly ultrasonographic evaluation. Horses were euthanatized at 6 weeks. Gross and histologic evaluation of tendon structure, fiber alignment, and collagen typing were used to define tendon architecture. Biochemical and molecular analyses of collagen, DNA, and proteoglycan and gene expression of collagen type I and type III, decorin, cartilage oligomeric matrix protein (COMP), and insulin-like growth factor-I were performed. RESULTS: Ultrasonography revealed no difference in rate or quality of repair between groups. Histologic evaluation revealed a significant improvement in tendon fiber architecture; reductions in vascularity, inflammatory cell infiltrate, and collagen type III formation; and improvements in tendon fiber density and alignment in ADNC-treated tendons. Repair sites did not differ in DNA, proteoglycan, or total collagen content. Gene expression of collagen type I and type III in treated and control tendons were similar. Gene expression of COMP was significantly increased in ADNC-injected tendons. CONCLUSIONS AND CLINICAL RELEVANCE: ADNC injection improved tendon organization in treated tendons. Although biochemical and molecular differences were less profound, tendons appeared architecturally improved after ADNC injection, which was corroborated by improved tendon COMP expression. Use of ADNC in horses with tendinitis appears warranted.     

Effects of phenylbutazone on bone activity and formation in horses

OBJECTIVE: To determine the effects of phenylbutazone (PBZ) on bone activity and bone formation in horses. ANIMALS: 12 healthy 1- to 2-year-old horses. PROCEDURES: Biopsy was performed to obtain unicortical bone specimens from 1 tibia on day 0 and from the contralateral tibia on day 14. Fluorochromic markers were administered IV 2 days prior to and on days 0, 10, 15, and 25 after biopsy was performed. Six horses received PBZ (4.4 mg/kg of body weight, PO, q 12 h) and 6 horses were used as controls. All horses were euthanatized on day 30 and tissues from biopsy sites, with adjacent cortical bone, were collected. Osteonal density and activity, mineral apposition rate (MAR), and percentage of mineralized tissue filling the biopsy-induced defects in cortical bone were assessed. Serum samples from all horses were analyzed for bone-specific alkaline phosphatase activity and concentration of PBZ. RESULTS: MAR was significantly decreased in horses treated with PBZ. Regional acceleratory phenomenon was observed in cortical bone in both groups but was significantly decreased in horses treated with PBZ. Osteonal activity was similar at all time points in all horses. In control horses, percentage of mineralized tissue filling the cortical defects was significantly greater in defects present for 30 days, compared with defects present for 14 days. Differences in percentage of mineralized tissue were not detected in horses treated with PBZ. CONCLUSIONS AND CLINICAL RELEVANCE: PBZ decreased MAR in cortical bone and appeared to decrease healing rate of cortical defects in horses.