Chondrogenic differentiation potential of adult and fetal equine cell types.

OBJECTIVE: To determine the chondrogenic potential of cells derived from interzone tissue, the normal progenitor of articular cartilage during fetal development, compared to that of adult bone marrow-derived and adipose-derived mesenchymal cell isolates. The objective of this study was to compare the chondrogenic potential of fetal musculoskeletal progenitor cells to adult cell types, which are currently used therapeutically to facilitate joint cartilage repair in equine clinical practice. The hypothesis tested was that cells derived from interzone tissue have a chondrogenic potential that exceeds that of adult bone marrow-derived and adipose-derived mesenchymal cell isolates. STUDY DESIGN: In vitro study. ANIMALS: Six young adult horses (15-17 months of age) and 6 equine fetuses aged 45-46 days of gestation. METHODS: Three-dimensional pellet cultures were established under chondrogenic conditions with fresh, primary cells isolated from adult (articular cartilage, bone marrow, adipose, dermis) and fetal (interzone, skeletal anlagen cartilage, dermis) tissues. Cellular morphology, pellet architecture, and proteoglycan synthesis were assessed in the pellet cultures. Steady state levels of ACAN (aggrecan core protein), COL2A1 (collagen type II), and COL1A1 (collagen type I) messenger RNA (mRNA) were compared among these cell types as pellet cultures and monolayer cultures. RESULTS: Adult articular chondrocytes, fetal interzone cells, and fetal anlage cells generated the largest pellets under these chondrogenic culture conditions. Pellets derived from adult articular chondrocytes and fetal anlage cells had the highest scores on a neocartilage grading scale. Fetal anlage and adult articular chondrocyte pellets had low steady-state levels of COL1A mRNA but high COL2A1 expression. Anlage chondrocyte pellets also had the highest expression of ACAN. CONCLUSION: Adult articular chondrocytes, fetal interzone cells, and fetal anlage chondrocytes exhibited the highest chondrogenic potential. In this study, adult adipose-derived cells exhibited very limited chondrogenesis, and bone marrow-derived cells had limited and variable chondrogenic potential. CLINICAL SIGNIFICANCE: Additional investigation of the high chondrogenic potential of fetal interzone cells and anlage chondrocytes to advance cell-based therapies in diarthrodial joints is warranted.

The Effects of Intra-Articular Triamcinolone and Autologous Protein Solution on Metabolic Parameters in Horses.

Intra-articular corticosteroids are a popular treatment choice for joint-associated pain and inflammation in horses despite recent work on the metabolic effects of these drugs. The goal of this project was to compare metabolic effects between intra-articular (IA) triamcinolone acetonide (TA) and an autologous protein solution (APS). Five mixed-breed geldings (4-9 years) were utilized for this project. Three identical and consecutive 28-day treatment blocks were used, with metacarpophalangeal IA treatments consisting of equal volumes of saline, a commercially available APS, or 9 mg of TA. Regular plasma and serum samples were collected for ACTH, cortisol, glucose, insulin, and thyroid hormone analysis, in addition to thyrotropin-releasing hormone (TRH) and oral sugar tests (OSTs). Significant treatment effects of IA TA were present at 48 h post-injection in both the TRH and the OST. There was also significant suppression by IA TA of baseline ACTH and cortisol between 2 h and 96 h post-treatment, hyperglycemia between 12 h and 48 h, and hyperinsulinemia at 32 h post-treatment. There were no treatment effects with respect to any measured thyroid hormones, nor were there any significant treatment effects of APS noted. Results suggest at least 2 days and up to 7 days should elapse between a single 9 mg IA TA treatment and OST and/or TRH testing. This study found that TA exhibits significant effects on ACTH, cortisol, glucose, and insulin, while the APS does not.

Identification of equine mares as reservoir hosts for pathogenic species of Leptospira.

Equine leptospirosis can result in abortion, stillbirth, neonatal death, placentitis, and uveitis. Horses can also act as subclinical reservoir hosts of infection, which are characterized as asymptomatic carriers that persistently excrete leptospires and transmit disease. In this study, PCR and culture were used to assess urinary shedding of pathogenic Leptospira from 37 asymptomatic mares. Three asymptomatic mares, designated as H2, H8, and H9, were PCR-positive for lipL32, a gene specific for pathogenic species of Leptospira. One asymptomatic mare, H9, was culture-positive, and the recovered isolate was classified as L. kirschneri serogroup Australis serovar Rushan. DNA capture and enrichment of Leptospira genomic DNA from PCR-positive, culture-negative samples determined that asymptomatic mare H8 was also shedding L. kirschneri serogroup Australis, whereas asymptomatic mare H2 was shedding L. interrogans serogroup Icterohaemorrhagiae. Sera from all asymptomatic mares were tested by the microscopic agglutination test (MAT) and 35 of 37 (94.6%) were seropositive with titers ranging from 1:100 to 1:3200. In contrast to asymptomatic mares, mare H44 presented with acute spontaneous abortion and a serum MAT titer of 1:102,400 to L. interrogans serogroup Pomona serovar Pomona. Comparison of L. kirschneri serogroup Australis strain H9 with that of L. interrogans serogroup Pomona strain H44 in the hamster model of leptospirosis corroborated differences in virulence of strains. Since lipopolysaccharide (LPS) is a protective antigen in bacterin vaccines, the LPS of strain H9 (associated with subclinical carriage) was compared with strain H44 (associated with spontaneous abortion). This revealed different LPS profiles and immunoreactivity with reference antisera. It is essential to know what species and serovars of Leptospira are circulating in equine populations to design efficacious vaccines and diagnostic tests. Our results demonstrate that horses in the US can act as reservoir hosts of leptospirosis and shed diverse pathogenic Leptospira species via urine. This report also details the detection of L. kirschneri serogroup Australis serovar Rushan, a species and serotype of Leptospira, not previously reported in the US.

Effect of Skeletal Paracrine Signals on the Proliferation of Interzone Cells.

OBJECTIVE: Articular cartilage in mammals has limited intrinsic capacity to repair structural defects, a fact that contributes to the chronic and progressive nature of osteoarthritis. In contrast, Mexican axolotl salamanders have demonstrated the remarkable ability to spontaneously and completely repair large joint cartilage lesions, a healing process that involves interzone cells in the intraarticular space. Furthermore, interzone tissue transplanted into skeletal defects in the axolotl salamander demonstrates a multi-differentiation potential. Cellular and molecular mechanisms of this repair process remain unclear. The objective of this study was to examine whether paracrine mitogenic signals are an important variable in the interaction between interzone cells and the skeletal microenvironment. DESIGN: The paracrine regulation of the proliferation of equine interzone cells was evaluated in an in vitro co-culture system. Cell viability and proliferation were measured in equine fetal interzone cells after exposure to conditioned medium from skeletal and nonskeletal primary cell lines. Steady-state expression was determined for genes encoding 37 putative mitogens secreted by cells that generated the conditioned medium. RESULTS: All experimental groups of conditioned media elicited a mitogenic response in interzone cells. Fetal anlage chondrocytes (P < 0.0001) and dermal fibroblasts (P < 0.0001) conditioned medium showed a significantly higher mitogenic potential compared with interzone cells. Conditioned medium from bone marrow-derived cells elicited a significantly higher proliferative response relative to that from young adult articular chondrocytes (P < 0.0001) or dermal fibroblasts (P < 0.0001). Sixteen genes had expression patterns consistent with the functional proliferation assays. CONCLUSIONS: The results indicate a mitogenic effect of skeletal paracrine signals on interzone cells.

Primary and secondary limb cellulitis in horses: 44 cases (2000-2006).

OBJECTIVE: To determine historical, physical, and microbiologic findings in horses with limb cellulitis and identify factors associated with short- and long-term outcome. DESIGN: Retrospective case series. ANIMALS: 44 horses with limb cellulitis. PROCEDURES: Information obtained from medical records included use, history, affected limb, diagnostic procedures, treatment, and short-term outcome. Long-term follow-up information was obtained by means of a telephone survey. RESULTS: Twenty-four horses had primary cellulitis, and 18 of the 24 (75%) had a hind limb affected. Results of microbiologic culture were positive for all 20 horses with primary cellulitis from which specimens were obtained, with coagulase-positive Staphylococcus spp recovered from 12 of the 20. Short-term survival rate was 67% (16/24) for horses with primary cellulitis; 7 of the 9 horses available for long-term follow-up were being used for their intended use, and 4 had had a recurrence. Results of microbiologic culture were positive for 13 of the 16 (81%) horses with secondary cellulitis from which specimens were obtained. Short-term survival rate for horses with secondary cellulitis was 90% (18/20). Eleven of the 17 horses available for long-term follow-up were being used for their intended use; 2 had had a recurrence. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that cellulitis can be a life-threatening condition in horses. Horses that were febrile at admission or that developed laminitis were significantly less likely to survive. The prognosis for return to function was guarded, and recurrence was a potential concern.

Surgical and traumatic wound infections, cellulitis, and myositis in horses.

Surgical site infections (SSIs) and traumatic wound management remain challenging clinical scenarios. The prevention of SSIs involves meticulous surgical technique and aftercare. Traumatic wounds require thorough evaluation to assess the involvement of synovial structures and radiographs to check for fractures. Chronic wounds can require a biopsy and histologic evaluation to obtain a diagnosis, because many underlying pathologic processes grossly appear similar but different treatment regimens are required. Early recognition and diagnosis of cellulitis and myositis enable the rapid aggressive intervention necessary for a positive outcome. Any delay in diagnosis and treatment increases the complication and mortality rates and makes these conditions difficult to treat successfully.