Osteochondral regeneration of the femoral medial condyle by using a scaffold-free 3D construct of synovial membrane-derived mesenchymal stem cells in horses.

BACKGROUND: Medical interventions for subchondral bone cysts in horses have been extensively studied. This study investigated the regeneration of articular cartilage and subchondral bone with scaffold-free three-dimensional (3D) constructs of equine synovial membrane-derived mesenchymal stem cells (SM-MSCs) isolated from three ponies and expanded until over 1.0 × 107 cells at passage 2 (P2). RESULTS: SM-MSCs were strongly positive for CD11a/CD18, CD44, and major histocompatibility complex (MHC) class I; moderately positive for CD90, CD105, and MHC class II; and negative for CD34 and CD45 on flow cytometry and differentiated into osteogenic, chondrogenic, and adipogenic lineages in the tri-lineage differentiation assay. After culturing SM-MSCs until P3, we prepared a construct (diameter, 6.3 mm; height, 5.0 mm) comprising approximately 1920 spheroids containing 3.0 × 104 cells each. This construct was confirmed to be positive for type I collagen and negative for type II collagen, Alcian blue, and Safranin-O upon histological analysis and was subsequently implanted into an osteochondral defect (diameter, 6.8 mm; depth, 5.0 mm) at the right femoral medial condyle. The contralateral (left femoral) defect served as the control. At 3 and 6 months after surgery, the radiolucent volume (RV, mm3) of the defects was calculated based on multiplanar reconstruction of computed tomography (CT) images. Magnetic resonance (MR) images were evaluated using a modified two-dimensional MR observation of cartilage repair tissue (MOCART) grading system, while macroscopic (gross) and microscopic histological characteristics were scored according to the International Cartilage Repair Society (ICRS) scale. Compared to the control sites, the implanted defects showed lower RV percentages, better total MOCART scores, higher average gross scores, and higher average histological scores. CONCLUSIONS: Implantation of a scaffold-free 3D-construct of SM-MSCs into an osteochondral defect could regenerate the original structure of the cartilage and subchondral bone over 6 months post-surgery in horses, indicating the potential of this technique in treating equine subchondral bone cysts.

Primary sinonasal malignant melanoma with systemic metastasis in a non-gray horse.

A 27-y-old Anglo-Arabian gelding with bay coat color was presented with a swelling of the left maxillary region. Fenestration on the left maxilla revealed that the left maxillary sinus was filled with black-red tissue. A portion of the tissue was excised and diagnosed histologically as malignant melanoma. Genotyping of the STX17 gene for gray coat color revealed that the horse did not have the "gray" factor. The horse was euthanized ~3 mo after first presentation. During autopsy, a black-to-gray mass extended from the left nasal cavity to the surrounding paranasal sinus and invaded the hard palate, cribriform plate, and the cranial portion of the left olfactory bulb. Moreover, identical black nodules were present in lymph nodes from the mandible to the larynx, and in the spleen, liver, kidney, and adrenal glands. However, masses were not found in the skin, perineal region, or pelvic cavity. All of the black-to-gray nodules were malignant melanomas that were histologically identical to the initial biopsy; tumor emboli were also found in the kidney. Sinonasal mucosal melanoma is a rare disease in horses.

Isolation of equine peripheral blood stem cells from a Japanese native horse.

The sizes of Japanese native horses have drastically decreased, and protection of these populations is important for Japanese horse culture. Social trials as well as scientific attempts are necessary for maintaining the breed. Mesenchymal stem cells (MSCs) have potential as a cell source for various cell therapies. However, there have been no reports on MSCs of Japanese native horses. We aimed to isolate and characterize MSCs from a Japanese native horse, the Noma horse. Plastic-adherent and self-replicating cells were isolated from a Noma horse's peripheral blood (PB). The isolated cells had trilineage potential and a surface antigen of mesenchymal cells, so they fulfilled the minimal criteria of MSCs. Therefore, PB can be one source of MSCs for Japanese native horses.

Methicillin-resistant Staphylococcus aureus ulcerative keratitis in a Thoroughbred racehorse.

We report the first case of methicillin-resistant Staphylococcus aureus (MRSA) keratitis in a racehorse. A 5-year-old mare developed punctate keratitis after racing. The corneal ulcer continued to expand despite ophthalmic antimicrobial therapy. On day 6, a conjunctival graft surgery was performed. The mare was euthanized, following colitis and laminitis development on day 10. MRSA was isolated from the corneal swab taken at the time of euthanasia. Immunohistochemical analysis demonstrated gram-positive and anti-S. aureus monoclonal antibody-positive cocci infiltration of the corneal stroma; and a diagnosis of MRSA ulcerative keratitis was made. An ophthalmic antimicrobial against the isolated MRSA did not improve the ocular lesion. The MRSA strain was found to be staphylococcal cassette chromosome mec type II, a strain frequently isolated from humans in Japan.

A study of the distribution of color Doppler flows in the superficial digital flexor tendon of young Thoroughbreds during their training periods.

Aim of this study was to evaluate the relationships of exercise and tendon injury with Doppler flows appearing in the superficial digital flexor tendon (SDFT) of young Thoroughbreds during training periods. The forelimb SDFTs of 24 one- to two-year-old Thoroughbreds clinically free of any orthopaedic disorders were evaluated using grey-scale (GS) and color Doppler (CD) images during two training periods between December 2013 to April 2015. Twelve horses per year were examined in December, February, and April in training periods that began in September and ended in April. The SDFT was evaluated in 3 longitudinal images of equal lengths (labelled 1, 2, 3 in order from proximal to distal), and 6 transversal images separated by equal lengths (labelled 1A, 1B, 2A, 2B, 3A and 3B in order from proximal to distal) of the metacarpus using both GS and CD. The running (canter and gallop) distance for 1 month before the date of the ultrasonographic examinations was increased in December, February, and April in both of the two training periods. CD flows defined as rhythmically blinking or pulsatory colored signals were found in 56 of 864 (6.4%) transversal CD images, in 28, 12, 13, and 3 images of 1A, 1B, 2A and 2B, respectively, and in 7, 14, and 35 images captured in December, February, and April, respectively. There were no longitudinal or transversal GS images indicating injury in the SDFTs in either of the two training periods. The increase of CD flows in the proximal regions of the SDFT are possibly related to the increase of the running distance during the training periods of the one- to two-year-old Thoroughbreds. Because no injury was diagnosed in the SDFTs by GS images during the training periods, the increase of CD flows in the proximal parts of SDFT is not necessarily predictive of tendon injury in the near future during the training period of young Thoroughbreds.

Pituitary pars intermedia dysfunction (equine Cushing's disease) in a Thoroughbred stallion: a single report.

Equine pituitary pars intermedia dysfunction (PPID) generally occurs in older horses showing hirsutism, delayed molting, weight loss, polydipsia, polyuria, laminitis, and reproductive disorders (in broodmares), but there have been no reports on stallions. This report presents a case of a 21-year-old Thoroughbred stallion that developed hirsutism and experienced delayed molting. There were no abnormal findings for semen quality or the stallion's sexual desire. The horse was diagnosed with PPID based on dexamethasone suppression test and plasma levels of adrenocorticotropic hormone. It was then medicated with pergolide mesylate. Since the horse died due to humerus fracture, an autopsy was conducted, and pituitary adenoma was confirmed. No pathological findings were defined in the testicles; therefore, reproductive activity might not have been impaired.

Mating behavior increases workload of the heart in Thoroughbred stallions.

To evaluate the influence of mating behavior on cardiac function, changes in heart rate (HR), electrocardiogram (ECG), hematocrit (Hct) and serum concentration of alpha-atrial natriuretic peptide (alpha-ANP) were evaluated in 10 clinically sound Thoroughbred stallions before and after mating behavior. The stallions were submitted twice to experimental pseudomating in the same month in 2009 and 2010. Measurements and blood samples were collected at a stable before mating (baseline) and at a covering yard before and after mating. ECG was recorded by a Holter-ECG system. Arrhythmias were detected in 5 stallions before or after mating behavior. Minimum HR (HRmin), maximum HR (HRmax) and HR recorded when the stallions entered into yard (HRent) and ejaculated (HRejc) were 34.2 ± 3.7, 168.9 ± 14.2, 141.8 ± 35.3 and 142.6 ± 27.3 beats/min, respectively. Time from entrance into the yard to ejaculation (mating time; MT) ranged from 30 to 2,103 sec and was highly correlated with HRent (r=-0.82) and the time required for attaining HRmax after entrance into the yard (dT HRmax) (r=0.87). Hct and serum alpha-ANP concentration significantly increased after ejaculation (60.0 ± 3.2%, P<0.0001, and 1.54 ± 0.61 ng/ml, P=0.0353) compared with the baselines values (46.9 ± 4.4%, 1.40 ± 0.60 ng/ml). HRent and Hct were significantly higher in the stallions with an MT of less than 5 min (n=5) compared with those (n=5) with an MT of more than 5 min (P=0.0324 and P=0.0082). Mating behavior increases the workload of the heart in Thoroughbred stallions.

Analysis of cartilage oligomeric matrix protein (COMP) in synovial fluid, serum and urine from 51 racehorses with carpal bone fracture.

We investigated the relationship between cartilage oligomeric matrix protein (COMP) levels in synovial fluid (SF), serum and urine and the development of osteochondral damage and osteophyte (OP) formation following intraarticular fractures of the carpus in racehorses in order to assess the clinical usefulness of COMP as a diagnostic biomarker of developmental osteoarthritis (OA). Two monoclonal antibodies (mAb clones 2A11 and 3C8) raised against equine COMP were shown to be capable of detecting the molecule in serum and urine as well as SF. Fifty-one samples were obtained from 26 OP-positive (OP(+)) and 25 OP-negative (OP(-)) racehorses with carpal bone fracture, in whom OP was ascertained arthroscopically and radiographically. The COMP measurements obtained using the two mAbs were highly correlated with each other in SF, serum, or urine. Horses with OP(+) showed a significantly higher [urinary COMP (microg)]/[urinary creatinine (mg)] ratio (4.94 +/- 5.10 and 1.46 +/- 1.19, using mAbs 2A11 and 3C8, respectively) than OP(-) horses (2.80 +/- 1.72 and 0.93 +/- 0.49, respectively). The relationship between serum and urine COMP levels and the period from injury to surgery were extrapolated using a polynomial expression. Measurement of COMP, especially in urine, has potential as a predictive marker of advanced OA following carpal bone fractures in racehorses.