In vitro chondrotoxicity of bupivacaine, levobupivacaine and ropivacaine and their effects on caspase activity in cultured canine articular chondrocytes.

Bupivacaine, levobupivacaine and ropivacaine are potent, long acting, amide-type local anesthetics that have several clinical applications including intra-articular administration. The objectives of this study were to evaluate their in vitro effects on cell viability and caspase activity to elucidate whether they activate the extrinsic or intrinsic pathways of apoptosis in canine articular chondrocytes. Chondrocytes in monolayer culture were treated with culture medium as the control, or with 0.062% (0.62 mg/mL) bupivacaine, 0.062% levobupivacaine, and 0.062% ropivacaine for 24 hr. Cell viability was evaluated using the live/dead, 3-(4,5-dimehylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), and Cell Counting Kit-8 (CCK-8) assays. Evaluation of caspase-3, caspase-8, and caspase-9 activity was performed using colorimetric assays. The MTT and CCK-8 assays were used to evaluate the effect of caspase inhibitors on local anesthetic chondrotoxicity. All three local anesthetics decreased chondrocyte viability after 24 hr (P<0.001). Apoptosis was induced through both the extrinsic and intrinsic pathways. Bupivacaine increased caspase-3, caspase-8, and caspase-9 activity (P<0.001). Levobupivacaine increased caspase-3 (P=0.03) while ropivacaine did not significantly upregulate activity for all three caspases. Caspase inhibition did not suppress bupivacaine chondrotoxicity whereas inhibition of caspase-8 and caspase-9 decreased ropivacaine chondrotoxicity and mildly attenuated levobupivacaine chondrotoxicity. In summary, the level of chondrotoxicity, the type of caspase activated, the level of caspase activation, and the response to caspase inhibitors was dependent on the type of local anesthetic. Therefore, ropivacaine may be a safer choice for intra-articular administration compared to levobupivacaine and bupivacaine.

Postmortem detection and histopathological features of canine spirocercosis-induced putative esophageal chondrosarcoma.

AIM: The objective of this study was to describe and characterize the postmortem and histopathological findings of putative esophageal chondrosarcoma associated with Spirocerca lupi. MATERIALS AND METHODS: Spirocerca-associated esophageal nodules were collected from 54 dogs at postmortem examination and were stained with hematoxylin and eosin. Of the cases examined, 15 were selected randomly for further investigation, of which 11 were classified as non-neoplastic nodules while 4 had changes reflecting a neoplastic process. RESULTS: In all four neoplastic cases, the wall of the esophageal nodules contained islands and nests of highly proliferative atypical chondroblasts within a cartilaginous matrix. However, there was no statistically significant association between gender (p=0.228), age (p=0.568), and breeds (p>0.05) with the occurrence of spirocercosis. Moreover, all esophageal nodules identified were located near the caudal segment, and their diameters ranged from 1 to 6 cm (4.7±1.5 cm). A number of worms in each nodule varied from 5 to 25 (11.3±5). CONCLUSION: Histopathology and cytology revealed that the wall of the esophageal nodules contained islands and nests of highly proliferative atypical chondroblasts within a cartilaginous matrix, a rare finding, and clinical challenge in spirocercosis.

Inhibitory effects of sodium pentosan polysulfate on formation and function of osteoclasts derived from canine bone marrow.

BACKGROUND: Sodium pentosan polysulfate (NaPPS) was testified as a chondroprotective drug in with a detailed rationale of the disease-modifying activity. This study was undertaken to determine whether anti-osteoarthritis drug, NaPPS inhibited osteoclasts (OC) differentiation and function. Canine bone marrow mononuclear cells (n = 6) were differentiated to OC by maintaining with receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) for up to 7 days with the treatment of NaPPS at concentration of 0, 0.2, 1 and 5 µg/mL. Differentiation and function of OC were accessed using tartrate-resistant acid phosphate (TRAP) staining and bone resorption assay, while monitoring actin ring formation. Invasion and colocalization patterns of fluorescence-labeled NaPPS with transcribed gene in OC were monitored. Gene expression of OC for cathepsin K (CTK), matrix metallopeptidase-9 (MMP-9), nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), c-Fos, activator protein-1(AP-1) and carbonic anhydrase II was examined using real-time PCR. RESULTS: Significant inhibition of OC differentiation was evident at NaPPS concentration of 1 and 5 µg/mL (p < 0.05). In the presence of 0.2 to 5 µg/mL NaPPS, bone resorption was attenuated (p < 0.05), while 1 and 5 µg/mL NaPPS achieved significant reduction of actin ring formation. Intriguingly, fluorescence-labeled NaPPS invaded in to cytoplasm and nucleus while colocalizing with actively transcribed gene. Gene expression of CTK, MMP-9 and NFATc1 were significantly inhibited at 1 and 5 µg/mL (p < 0.05) of NaPPS whereas inhibition of c-Fos and AP-1 was identified only at concentration of 5 µg/mL (p < 0.05). CONCLUSIONS: Taken together, all the results suggest that NaPPS is a novel inhibitor of RANKL and M-CSF-induced CTK, MMP-9, NFATc1, c-Fos, AP-1 upregulation, OC differentiation and bone resorption which might be a beneficial for treatment of inflammatory joint diseases and other bone diseases associated with excessive bone resorption.

Differentiation potential of synoviocytes derived from joints with cranial cruciate ligament rupture and medial patella luxation in dogs.

The objective of this study was to assess the differentiation capability of synoviocytes derived from dogs with inflammatory joint conditions. Cranial cruciate ligament ruptured (CCLr) (n=12) and medial patella luxated (MPL) (n=10) knee joints of the dogs were used to collect the synovial membrane (SM). Synoviocytes were enzymatically released from the SM and analyzed by flow cytometry for specific cellular markers (CD44 and CD90) of mesenchymal stem cells (MSCs), while doing histopathology from another part of SM sections. Under specific culture conditions, synoviocytes were forced to differentiate into chondrogenesis, adipogenesis, osteogenesis and osteoclastogenesis to investigate the multipotency. Upon treatments phenotypes of cell cultures were analyzed by histopathology and by semi-quantitative reverse transcriptase polymerase chain reaction for the expression of each differentiation marker genes. Although flow cytometry showing similar MSCs populations in CCLr and MPL synovium, synovial cells derived from CCLr showed higher multipotency compared to MPL-derived samples. Further, synovial changes such as vascularity, mononuclear cell infiltration and cellular hypertrophy were more pronounced in CCLr-derived synovial tissue than in MPL. Taken together, these findings suggested that the differentiation capability of SM-derived multipotent stem cells varies with inflammatory severity occurring in different joint conditions.

Pentosan polysulfate inhibits IL-1ß-induced iNOS, c-Jun and HIF-1α upregulation in canine articular chondrocytes.

Osteoarthritic (OA) chondrocytes are shown to express inducible nitric oxide synthase (iNOS) which produces high concentrations of nitric oxide (NO), particularly when stimulated with proinflammatory cytokines. NO is involved in OA cartilage degradation. On the other hand, c-Jun N-terminal Kinase (JNK) pathway mediates the activation and transcription of c-Jun, which is required for interleukin-1 (IL-1)-induction of matrix metalloproteinases-13 (MMP-13) in OA pathogenesis. Therefore, the selective inhibition of iNOS and c-Jun is a promising target for treatment and prevention of OA. The purpose of the study was to investigate the inhibitory effects of pentosan polysulfate (PPS) on IL-1ß-induced iNOS, c-Jun and HIF-α isoforms upregulation in canine articular chondrocytes (CACs). Primary (P0) chondrocytes were isolated and cultured from femoral head cartilages of three (3) dogs. First passage (P1) chondrocytes were preincubated with 0, 1, 5, 15 and 40 µg/mL of PPS for 4 hr before treatment with 10 ng/mL rhIL-1ß for a further 8 hr. In addition, we evaluated the effects of single and multiple cytokine with or without LPS on iNOS protein induction. PPS significantly inhibited (P < 0.05) IL-1ß-induced iNOS, c-Jun and HIF-1α mRNA upregulation in a dose-dependent pattern. iNOS mRNA was significantly inhibited at 15 and 40 µg/mL whereas c-Jun and HIF-1α were significantly downregulated at 5, 15 and 40 µg/mL of PPS compared to chondrocytes treated with only rhIL-1ß. Intriguingly, CACs were recalcitrant to single IL-1ß, TNF-α or LPS-induction of iNOS protein including to a combination of IL-1ß+TNF-α, IL-1ß+LPS except to TNF-α+LPS and IL-1ß+TNF-α+LPS suggestive of a protective mechanism from iNOS detrimental effects on perpetuating OA. IL-1ß+TNF-α+LPS-induced iNOS protein expression was significantly abrogated by PPS. We demonstrate for the first time that PPS is a novel inhibitor of IL-1ß-induced iNOS, c-Jun, and HIF-1α mRNA upregulation and iNOS protein induction which may be beneficial for prevention and treatment OA.

Effects of pentosan polysulfate and polysulfated glycosaminoglycan on chondrogenesis of canine bone marrow-derived mesenchymal stem cells in alginate and micromass culture.

Mesenchymal stem cells (MSC) are a potential alternative source of differentiated chondrocytes for cartilage tissue regeneration and repair of osteoarthritic (OA) joints. We investigated the effects of pentosan polysulfate (PPS) and polysulfated glycosaminoglycan (PSGAG) on chondrogenesis of canine bone marrow-derived mesenchymal stem cells (cBMSC) in alginate and micromass cultures (MMC). Chondrogenic differentiation medium (CDM) was supplemented with PPS or PSGAG at concentrations of 0 (positive control; PC), 1, 3 and 5 µg/ml. 10% DMEM was used as negative control. Chondrocyte phenotype was analyzed by quantitative real-time PCR (qPCR) for alginate cultures and Alcian blue staining for proteoglycan (PG) synthesis for MMC. In alginate culture, PPS and PSGAG showed no significant effect on type II collagen, aggrecan and HIF-2α mRNA expression. PPS had no significant effect on type I collagen whereas PSGAG significantly upregulated (P<0.05) it at all concentrations relative to other treatments. PPS demonstrated a dose-dependent inhibitory effect on type X collagen mRNA with significant inhibition observed at 5 µg/ml compared to the NC. PSGAG showed an inverse effect on type X collagen with 1 µg/ml significantly inhibiting its expression while increase in the concentration correspondingly increased type X collagen expression. In MMC, PPS significantly enhanced chondrogenesis and PG deposition whereas PSGAG inhibited chondrogenesis and promoted a fibrocartilage-like phenotype with reduced PG deposition. While PPS enhances chondrogenesis of cBMSC in MMC, the response of MSC to chondroinductive factors is culture system-dependent and varies significantly between alginate and MMC.