Characterization of normal canine anterior cruciate ligament-associated synoviocytes.

This study was designed to identify and quantify synoviocyte phenotypes enveloping the canine anterior cruciate ligament (ACL) to test the hypothesis that there are at least two synoviocyte phenotypes, each with distinct quantities and topographical distributions. CD18 and HSP25 epitopes were colocalized in the synovium of 10 normal canine ACLs. Sagittal sections were prepared from medial, central, and lateral aspects of each ACL and phenotypes were quantified in the proximal, middle, and distal aspects of each section. Distinct synoviocyte populations stained positive for CD18 (CD18+) or HSP25 (HSP25+), and a small population of cells stained for both epitopes (DS+). The proportion (mean +/- SEM) of HSP25+ synoviocytes (57% +/- 7.5%) was significantly greater than the proportion of CD18+ synoviocytes (27% +/- 8.2%), which was significantly greater than the proportion of DS+ synoviocytes (16% +/- 3.5%). Reverse transcriptase polymerase chain reaction (RT-PCR), Western blot analysis, and immunoelectron microscopy confirmed the presence of CD18 and HSP25 epitopes in the canine ACL. Identification and quantification of ACL synoviocytes may serve as the foundation for future studies involving ACL disease or reconstruction.

Comparison of chondrogenic potential in equine mesenchymal stromal cells derived from adipose tissue and bone marrow.

OBJECTIVE: To compare the chondrogenic potential of adult equine mesenchymal stem cells derived from bone marrow (MSCs) or adipose tissue (ASCs). STUDY DESIGN: In vitro experimental study. ANIMALS: Adult Thoroughbred horses (n=11). METHODS: BM (5 horses; mean [+/-SD] age, 4+/-1.4 years) or adipose tissue (6 horses; mean age, 3.5+/-1.1 years) samples were obtained. Cryopreserved MSCs and ASCs were used for pellet cultures in stromal medium (C) or induced into chondrogenesis+/-transforming growth factor-3 (TGFbeta(3)) and bone morphogenic factor-6 (BMP-6). Pellets harvested after 3, 7, 14, and 21 days were examined for cross-sectional size and tissue composition (hematoxylin and eosin), glycosaminoglycan (GAG) staining (Alcian blue), collagen type II immunohistochemistry, and by transmission electron microscopy. Pellet GAG and total DNA content were measured using dimethylmethylene blue and Hoechst DNA assays. RESULTS: Collagen type II synthesis was predominantly observed in MSC pellets from Day 7 onward. Unlike ASC cultures, MSC pellets had hyaline-like matrix by Day 14. GAG deposition occurred earlier in MSC cultures compared with ASC cultures and growth factors enhanced both MSC GAG concentrations (P<.0001) and MSC pellet size (P<.004) after 2 weeks in culture. CONCLUSION: Equine MSCs have superior chondrogenic potential compared with ASCs and the equine ASC growth factor response suggests possible differences compared with other species. CLINICAL RELEVANCE: Elucidation of equine ASC and MSC receptor profiles will enhance the use of these cells in regenerative cartilage repair.

Fully 2'-deoxy-2'-fluoro substituted nucleic acids induce RNA interference in mammalian cell culture.

RNA interference is a phenomenon in which RNA molecules elicit potent and sequence-specific post-transcriptional gene silencing. Recent studies have shown that small interfering RNA containing pyrimidine 2'-fluoro modifications elicit RNAi. In this study, we demonstrate that fully-2'-fluorinated nucleic acids can be generated for RNAi studies through either custom solid-phase synthesis or in vitro transcription using a mutated polymerase and fluorinated nucleoside triphosphates. Single-stranded and hybridized fully-2'-fluorinated nucleic acids were subjected to a ribonuclease to assess their resistance to digestion. Duplex siFNA and antisense fully-2'-fluorinated nucleic acids were evaluated for their ability to knockdown green fluorescent protein expression in mammalian cell culture. Based on the results, fully-2'-fluorinated nucleic acids can be successfully generated, and fully-2'-fluorinated nucleic acids products show superior resistance to digestion over native RNA. Melt curve analysis suggests that transcribed fully-2'-fluorinated nucleic acids may contain base miscoding errors or early termination products. Small interfering fluoronucleic acid can induce RNAi and the silencing efficiency is nearly equivalent to the unmodified small interfering RNA species. Silencing from antisense fully-2'-fluorinated nucleic acids was greatly reduced relative to the duplex form. The lack of silencing activity from single-stranded fully-2'-fluorinated nucleic acids, combined with reverse transcription polymerase chain reaction data showing that mRNA decreases following siFNA treatment, suggests that knockdown from siFNA is likely enzymatically driven as opposed to simple translational arrest.

Molecular identification of Phialophora oxyspora as the cause of mycetoma in a horse.

CASE DESCRIPTION: An 18-year-old mare was evaluated for an oral mass that developed after extraction of a broken incisor. CLINICAL FINDINGS: An ulcerated, firm, darkly pigmented, approximately 5-cm-diameter spherical mass involved the gingiva lateral and dorsal to the right first to third maxillary incisors. Osteolysis of the roots of the first and second right maxillary incisors and periosteal proliferation of the adjacent premaxilla margins were apparent on radiographs. Histologic examination of the mass revealed multiple coalescing and ramifying foci of abscess formation, each containing a well-defined, discrete, black mass (2 to 7 mm in diameter). Myriad fungal hyphae enmeshed in a black, granular, cementlike material were within each of the black structures. Mycetoma was the histologic diagnosis. The causative agent could not be identified via culture because of lack of distinguishing characteristics. Fungal DNA was isolated from frozen fungal cultures and paraffin sections. The D1/D2 domains of the large subunit P gene rDNA were amplified and sequenced. The sequences of the D1/D2 domains of both isolates were 96% homologous with those of Phialophora oxyspora. TREATMENT AND OUTCOME: The mass was surgically excised, the local area curetted, and the wound allowed to heal by second intention. Postoperative treatment consisted of administration of phenylbutazone and IV administration of sodium iodide followed by oral administration of potassium iodide. There was no evidence of recurrence 1 year later. CLINICAL RELEVANCE: Mycetomata should be a differential diagnosis for equine gingival masses. Identification of the fungal agent can be critical for selection of optimal treatments. Molecular methods may permit definitive identification when standard phenotypic-based identification criteria are inconclusive.

In vivo evaluation of intra-articular protection in a novel model of canine cranial cruciate ligament mid-substance elongation injury.

OBJECTIVES: To evaluate the effects of intra-articular protection (IAP) on the canine cranial cruciate ligament (CrCL) and stifle in a CrCL midsubstance elongation injury model. STUDY DESIGN: Experimental longitudinal cohort study. ANIMALS: Skeletally mature female mixed breed hounds (n=12; mean+/-SEM weight, 25.6+/-0.7 kg). METHODS: After CrCL elongation in 1 stifle of each dog, IAP was applied in 6 joints. In vivo assessment included radiographs, cranial-caudal joint translation, gait analysis, and synovial fluid levels of 3B3(-) (proteoglycan epitope) and C2C (collagen II neoepitope) up to 12 weeks after surgery. Joint translation and rotation were quantified at necropsy. CrCL midsubstance length was determined before and after elongation and at necropsy. CrCLs were subjectively assessed with light microscopy. Comparisons were made between stifles containing elongated CrCLs with and without IAP and unoperated controls. RESULTS: Four weeks after surgery, ground reaction forces were significantly decreased in operated limbs. Absolute C2C levels were significantly elevated in operated stifles 4 weeks post-surgery. C2C and 3B3(-) levels normalized to total protein were significantly elevated in IAP+ stifles 8 weeks after surgery. Protected CrCLs appeared to have decreased granulation tissue and better collagen fiber alignment. CONCLUSIONS: IAP has negligible effects on the canine stifle based on the response variables evaluated in this 12-week study. Protection of elongated CrCLs may promote reduced, organized scar formation. CLINICAL RELEVANCE: These results support the healing capacity of the canine CrCL midsubstance following elongation injury and IAP application to potentially reduce cicatrix formation in elongated CrCLs.