In vitro biomechanical properties of sole tissues: Comparison between healthy and ulcerated bovine claws.

Sole ulcers are reportedly one of the most prevalent diseases associated with lameness in dairy cattle, significantly affecting animal welfare and farm profitability. The degree to which sole soft tissues, healthy or ulcerated, are able to maintain their structure and function when subjected to compressive forces remains unknown. Therefore, the aims of the present study were to assess sole tissue biomechanics in healthy and ulcerated claws and to describe correlated histology. Cylindrical samples were harvested from zones 4 and 6, as described by the international foot map, from hind lateral healthy (n = 12) and ulcerated bovine claws (n = 8; animals n = 12). Tissue biomechanics and morphology were evaluated via compressive tests and hematoxylin-eosin-phloxine-saffron staining, respectively. A 2-sample t-test was used to compare zones' mechanical properties between healthy and ulcerated tissues, and the Cochran-Mantel-Haenszel test was used to measure the effect of claw zone on histology. The fibril modulus (Ef) and permeability (k) respectively increased and decreased in ulcerated claws (Ef = 0.201 ± 0.104 MPa; k = 0.128 ± 0.069 mm2/MPa·s) compared with healthy claws (Ef = 0.105 ± 0.050 MPa; k = 0.452 ± 0.365 mm2/MPa·s) only for zone 6. Histology scores equal to or greater than 3 were associated with macroscopic presence of ulceration. A higher proportion of adipose tissue (30% or more) was associated with zone 6 compared with zone 4, but no difference was seen between healthy and ulcerated claws. Ulcerated claws had a higher prevalence of exostoses compared with healthy ones (33% vs. 8%). Sole soft tissues showed, as hypothesized, a viscoelastic behavior using unconfined compression testing, which, however, may not reflect in vivo loading conditions. Clinical and histological signs of sole ulceration were not associated with decreased strength of the supportive apparatus of the distal phalanx in zone 4 in this study.

A cross-sectional survey on wounds in horses in New Zealand.

AIM: To determine the frequency and type of skin wounds encountered by New Zealand veterinarians in their equine patients, the duration and estimated costs of treatment as well as the expected outcomes for these wounds. METHODS: An online survey was sent to all veterinarians registered with the New Zealand Equine Veterinary Association. The survey comprised questions on the location and experience of respondents, the number of wound-related cases in relation to the total equine caseload, the type and anatomical location of wounds treated, the frequency, duration and costs of treatments, the outcome of wound treatment and an estimate of the most common causes of death or euthanasia in their equine patients. RESULTS: The survey response rate was 110/262 (41.9%). The median number of equine cases seen by respondents was 20 (interquartile range (IQR) 6-60) per month; of these, five (IQR 2-10) were wound related. Wounds ranked third after lameness and respiratory disease for the relative frequency with which respondents encountered them. Of 102 respondents 59 (58%) reported that their clients frequently treated wounds incurred by their horse without consulting a veterinarian. Wounds on the distal limb, whether involving only the skin or also deeper structures, were reported by 86/101 (85%) respondents as the most frequently encountered. Wounds in this location also incurred the longest treatment period and were the most prone to develop complications. Finally, wounds ranked second, after colic, as the most common cause of death/euthanasia in the veterinary respondents' equine patients. CONCLUSIONS: The data obtained via the survey indicate that skin wounds, particularly on the distal limb, are a common occurrence in horses in New Zealand and, when they involve structures underlying the skin, are costly and time-consuming to manage and may lead to decreased performance, retirement or euthanasia. Consequently, we recommend that more effort be devoted to the education of equine veterinarians and owners, and that appropriate research funds be allocated to help improve patient outcomes.

Osteopontin expression in healing wounds of horses and in human keloids.

REASONS FOR PERFORMING STUDY: Convincing evidence shows that persistent or excessive expression of osteopontin (OPN) is linked to fibroproliferation of various organs in laboratory animals and in man, such that its downregulation is a logical therapeutic objective. OBJECTIVES: To investigate OPN expression in an equine model of wound healing and in clinical specimens of equine exuberant granulation tissue and human keloids in an effort to better understand the contribution of this protein to inflammation-associated skin fibrosis. STUDY DESIGN: Description of gene and protein expression in an experimental equine model of wound healing and clinical specimens in horse and man. METHODS: Osteopontin gene expression was evaluated by quantitative PCR, while protein expression was investigated by means of immunohistochemical staining. RESULTS: Quantitative PCR showed that the OPN gene is expressed in normal intact skin of horses and continues to be expressed during the wound-healing process. An increase in gene expression was observed throughout the phases of wound healing, with a final decrease at wound closure. The protein was not detected in normal skin. Keratinocytes in wound-edge samples did not express the protein, whereas dermal immunoreactivity was confined to inflammatory cells. Healed wounds were devoid of staining. Equine exuberant granulation tissue showed immunoreactivity of the surrounding epidermis, infiltrating neutrophils, mononuclear cells, endothelial cells and fibroblasts. Human keloids showed OPN immunoreactivity throughout the epidermis as well as in mononuclear cells and scattered fibroblasts. CONCLUSIONS: Immunohistochemical data show a different pattern of expression between normally healing and fibrotic wounds (exuberant granulation tissue and keloids), thus suggesting a role in fibroproliferation in horses and man.

Differentiation of equine induced pluripotent stem cells into a keratinocyte lineage.

REASONS FOR PERFORMING STUDY: Skin trauma in horses often leads to the development of chronic nonhealing wounds that lack a keratinocyte cover, vital for healing. Reports in mouse and man confirm the possibility of generating functional keratinocytes from induced pluripotent stem cells (iPSC), thus presenting myriad potential applications for wound management or treatment of skin disease. Similarly, differentiation of equine iPSC (eiPSC) into a keratinocyte lineage should provide opportunities for the advancement of veterinary regenerative medicine. OBJECTIVES: The purpose of this study was to develop an efficient method for the differentiation of eiPSC into a keratinocyte lineage. It was hypothesised that eiPSC can form differentiated keratinocytes (eiPSC-KC) comparable with primary equine keratinocytes (PEK) in their morphological and functional characteristics. STUDY DESIGN: Experimental in vitro study. METHODS: Equine iPSC established using a nonviral system were treated for 30 days with retinoic acid and bone morphogenetic protein-4 to induce directed differentiation into iPSC-KC. Temporospatial gene and protein expression by eiPSC-KC was measured at weekly intervals of differentiation and in response to calcium switch. Proliferative and migratory capacities of eiPSC-KC were compared with those of PEK. RESULTS: Equine iPSC, upon directed differentiation, showed loss of pluripotency genes and progressive increase in pancytokeratin expression indicating ectodermal specification into keratinocytes. High differentiation efficiency was achieved, with 82.5% of eiPSC expressing keratin 14, a marker of epidermal-specific basal stem cells, after 30 days of directed differentiation. Moreover, the proliferative capacity of eiPSC-KC was superior, while the migratory capacity (measured as the ability to epithelise in vitro wounds) was comparable with that of PEK. CONCLUSIONS: This proof of concept study suggests that eiPSC can successfully be differentiated into equine keratinocytes (eiPSC-KC) with features that are promising to the development of a stem cell-based skin construct, with the potential to regenerate lost or damaged skin.

Imaging and histological features of central subchondral osteophytes in racehorses with metacarpophalangeal joint osteoarthritis.

REASONS FOR PERFORMING STUDY: Marginal osteophytes represent a well known component of osteoarthritis in man and animals. Conversely, central subchondral osteophytes (COs), which are commonly present in human knees with osteoarthritis, have not been reported in horses. OBJECTIVES: To describe and compare computed radiography (CR), single-slice computed tomography (CT), 1.5 Tesla magnetic resonance imaging (MRI), and histological features of COs in equine metacarpophalangeal joints with macroscopic evidence of naturally-occurring osteoarthritis. METHODS: MRI sequences (sagittal spoiled gradient recalled echo [SPGR] with fat saturation, sagittal T2-weighted fast spin echo with fat saturation [T2-FS], dorsal and transverse T1-weighted gradient-recalled echo [GRE], and sagittal T2*-weighted gradient echo with fast imaging employing steady state acquisition [FIESTA]), as well as transverse and reformatted sagittal CTI and 4 computed radiographic (CR) views of 20 paired metacarpophalangeal joints were acquired ex vivo. Following macroscopic evaluation, samples were harvested in predetermined sites of the metacarpal condyle for subsequent histology. The prevalence and detection level of COs was determined for each imaging modality. RESULTS: Abnormalities consistent with COs were clearly depicted on MRI, using the SPGR sequence, in 7/20 (35%) joints. They were identified as a focal hypointense protuberance from the subchondral plate into the cartilage, at the palmarodistal aspect (n=7) and/or at the very dorsal aspect (n=2) of the metacarpal condyle. COs were visible but less obvious in 5 of the 7 joints using FIESTA and reformatted sagittal CT, and were not identifiable on T2-FS, T1-GRE or CR. Microscopically, they consisted of dense bone protruding into the calcified cartilage and disrupting the tidemarks, and they were consistently associated with overlying cartilage defects. CONCLUSIONS: Subchondral osteophytes are a feature of osteoarthritis of equine metacarpophalangeal joints and they may be diagnosed using 1.5 Tesla MRI and CT. POTENTIAL RELEVANCE: Central subchondral osteophytes on MRI represent indirect evidence of cartilage damage in horses.

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.

Expression of transforming growth factor beta(1), beta(3), and basic fibroblast growth factor in full-thickness skin wounds of equine limbs and thorax.

OBJECTIVE: To map the expression of transforming growth factor (TGF)-beta(1), TGF-beta(3), and basic fibroblast growth factor (bFGF) in full-thickness skin wounds of the horse. To determine whether their expression differs between limbs and thorax, to understand the pathogenesis of exuberant granulation tissue. STUDY DESIGN: Six wounds were created on one lateral metacarpal area and one midthoracic area of each horse. Sequential wound biopsies allowed comparison of the temporal expression of growth factors between limb and thoracic wounds. ANIMALS: Four 2- to 4-year-old horses. METHODS: Wounds were assessed grossly and histologically at 12 and 24 hours, and 2, 5, 10, and 14 days postoperatively. ELISAs were used to measure the growth factor concentrations of homogenates of wound biopsies taken at the same timepoints. RESULTS: TGF-beta(1) peaked at 24 hours in both locations and returned to baseline in thoracic wounds by 14 days but remained elevated in limb wounds for the duration of the study. Expression kinetics of TGF-beta(3) differed from those of TGF-beta(1). TGF-beta(3) concentrations gradually increased over time, showing a trend toward an earlier and higher peak in thoracic compared with limb wounds. bFGF expression kinetics resembled those of TGF-beta(1), but no statistically significant differences existed between limb and thoracic wounds. CONCLUSIONS: Growth factor expression is up-regulated during normal equine wound repair. TGF-beta(1) and TGF-beta(3) show a reciprocal temporal regulation. Statistically significant differences exist between limb and thoracic wounds with respect to TGF-beta(1) expression. CLINICAL RELEVANCE: The persistence of TGF-beta(1) expression in leg wounds may be related to the development of exuberant granulation tissue in this location, because TGF-beta(1) is profibrotic.

Incomplete nasomaxillary dysplasia in a foal.

Atresia of the nasal punctum is the most common congenital anomaly for the equine nasolacrimal system. Nasomaxillary dysplasia has not been previously documented in foals, is of unknown etiology, and appears to be a rare condition. Conjunctivomaxillary sinostomy was successful in resolving the epiphora.

Repair and function of synovium after arthroscopic synovectomy of the dorsal compartment of the equine antebrachiocarpal joint.

The reparative ability of equine synovium was determined by gross, histological, and ultrastructural examination. The functional potential of the synovium was estimated by examination of synovial cell organelles with transmission electron microscopy. Results from rested and exercised horses were compared to determine the effect of exercise on synovial healing. The response of synovectomized joint to exercise was evaluated with a standardized lameness examination and by gross, histological, and histochemical observations of the articular cartilage. A 7-mm diameter motorized synovial resector was used to perform a subtotal synovectomy in 1 antebrachiocarpal joint of each of 8 horses; the contralateral joint served as a control. After 2 months rest, four randomly selected horses were rigorously exercised for the remainder of the study; the other four horses continued paddock rest. Lameness examinations and synovial fluid analyses were conducted at 0, 2, 30, 60, and 120 days. Synovium and articular cartilage from all horses were examined at necropsy at 120 days. None of the horses were lame during the study, and transient synovitis occurred in the synovectomized joints. The hyaluronan concentration of treated joints decreased at 2 days but returned to normal by 60 days. Synovial fluid composition, including hyaluronan concentration, was unchanged by exercise. Significant cartilage damage was not observed in any of the joints. At 120 days, the healing synovium was devoid of villi and its subintima was fibrotic, however transmission electron microscopy confirmed that an intimal layer was present within the repair tissue. The cells within the repair tissue appeared actively engaged in both synthesis and phagocytosis. Exercise did not modify any of these findings. The results of this study suggest that 120 days after subtotal synovectomy, the joint environment was maintained and and the resected synovium had evidence of restoration and increased metabolic potential. Synovectomized joints withstood exercise but synovial repair was not accelerated by exercise.