Angela Y. Au

Consil bioactive glass particles enhance osteoblast proliferation and selectively modulate cell signaling pathways in vitro.

Consil Bioglass is a commercially available bioactive glass formulation previously shown in clinical studies to support osteogenesis and the repair of bony defects in dogs and cats. Previous in vitro studies confirm that Consil particles are able to bond directly with bone while promoting osteoblast proliferation and extracellular matrix production. However, the cellular mechanisms mediating their clinical effect remain unclear. This study evaluated whether enhancement of osteoblast proliferation by Consil particles is associated with signal transduction. Consil particles maintained the osteoblast phenotype and enhanced proliferation of canine osteoblasts for up to 21 days in culture. Stimulation of proliferation and maintenance of phenotype expression were accompanied by the modulation of selective cell signaling pathways including integrins, the mitogen-activated protein kinases (MAPKs), and the immediate-early gene c-Jun. These genes have been documented to mediate osteoblast growth and differentiation. The signal transduction occurs in a time-dependent manner in which Consil particles induce a decrease in the pattern of MAPK and c-Jun gene transcription from 4 to 24 h and a subsequent return to control levels by 7 days in culture. Our observations suggest that Consil Bioglass particles may provide cues that enhance cell division necessary for facilitating bone regeneration and the repair of bony defects.

Inhibition of cytokine expression and prostaglandin E2 production in monocyte/macrophage-like cells by avocado/soybean unsaponifiables and chondroitin sulfate.

Osteoarthritis is characterized by inflammation and increased production of pro-inflammatory mediators including cytokines and prostaglandin E2 (PGE2). Macrophage-like cells in synovial tissue produce these mediators which induce degradative enzymes that break down cartilage. We determined whether avocado/soybean unsaponifiables (ASU) and chondroitin sulfate (CS) can inhibit cytokine expression and PGE2 production using monocyte/macrophage-like cell models. Cells were incubated for 24 hours with either control media alone, ASU alone (NMX1000; 8.3 ?g/ml), CS alone (TRH122; 20 ?g/ml), or a combination of both preparations. Cells were activated with cytokines or lipopolysaccharide for 1 or 24 hours to determine cytokine gene expression by RT-PCR and PGE2 production by immunoassay, respectively. In response to activation, THP-1 cells exhibited increased expression of TNF-? and IL-1?, while RAW cells increased synthesis of PGE2. Cytokine expression and PGE2 synthesis were significantly decreased by the combination of ASU and CS compared to the individual treatments alone (P<0.05). Our finding supports the potential of the ASU and CS combination to suppress the synthesis of pro-inflammatory mediators in the joint.

Modulation of inflammation and oxidative stress in canine chondrocytes

OBJECTIVE To determine whether oxidative stress could be induced in canine chondrocytes in vitro. SAMPLE Chondrocytes obtained from healthy adult mixed-breed dogs. PROCEDURES Harvested chondrocytes were maintained at 37°C with 5% CO2 for 24 hours. To assess induction of oxidative stress, 2 stimuli were used: hydrogen peroxide and a combination of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). To determine the effect of hydrogen peroxide, a set of chondrocyte-seeded plates was incubated with control medium alone or hydrogen peroxide (100, 200, or 300μM) for 24 hours. For inhibition of oxidative stress, cells were incubated for 24 hours with N-acetylcysteine (NAC; 10mM) before exposure to hydrogen peroxide. Another set of chondrocyte-seeded plates was incubated with control medium alone or with IL-1β (10 ng/mL) and TNF-α (1 ng/mL) for 24 hours. Supernatants were obtained for measurement of prostaglandin E2 production, and cell lysates were used for measurement of superoxide dismutase (SOD) activity and reduced-glutathione (GSH) concentration. RESULTS Chondrocytes responded to the oxidative stressor hydrogen peroxide with a decrease in SOD activity and GSH concentration. Exposure to the antioxidant NAC caused an increase in SOD activity in hydrogen peroxide-stressed chondrocytes to a degree comparable with that in chondrocytes not exposed to hydrogen peroxide. Similarly, NAC exposure induced significant increases in GSH concentration. Activation with IL-1β and TNF-α also led to a decrease in SOD activity and increase in prostaglandin E2 production. CONCLUSIONS AND CLINICAL RELEVANCE Canine chondrocytes responded to the oxidative stress caused by exposure to hydrogen peroxide and cytokines. Exposure to oxidative stress inducers could result in perturbation of chondrocyte and cartilage homeostasis and could contribute to the pathophysiology of osteoarthritis. Use of antioxidants, on the other hand, may be helpful in the treatment of arthritic dogs.

Micropatterned agarose scaffolds covalently modified with collagen for culture of normal and neoplastic hepatocytes.

Anchorage-dependent cells including hepatocytes, the main functional cellular constituent comprising liver tissue, require a substrate for cell adhesion when cultured outside their native tissue. The challenge with hepatocyte culture is that material substrates and designs supporting hepatocyte attachment, phenotype, and function are not readily available. Our laboratory previously published that type I collagen found in the liver extracellular matrix supports hepatocyte culture. We hypothesized that micropatterned agarose with a coating of collagen covalently bound to the surface would facilitate hepatocyte adhesion and phenotype. To test this hypothesis, primary canine hepatocytes and neoplastic human HepG2 hepatocellular carcinoma cells were cultured on these substrates. Hepatocyte adhesion was dependent on the cell type and also the micropattern design. Viable normal and neoplastic hepatocytes attached to the microchannel troughs rather than on the ridges. In contrast, hepatocyte adhesion on the microcircular patterns was similar to control agarose as cells did not sense differences in surface topology on these substrates. Neoplastic cells exhibited a distinct difference in growth behavior following 7 days in culture on the microchannel patterns, exhibiting aberrant proliferation relative to normal hepatocytes which did not proliferate. Our results suggest that patterned microchannel agarose may be useful to evaluate hepatoprotective and noxious agents.

Modulation of cytokine-induced prostaglandin E2 production in cultures of articular chondrocytes obtained from carpal joints of camels (Camelus dromedarius)

OBJECTIVE To determine whether camel articular chondrocytes can be maintained in tissue culture without phenotype loss and whether the response to cytokine stimulation can be modulated. SAMPLE POPULATION Cartilage from 4 carpal joints of healthy adult dromedary camels (Camelus dromedarius). PROCEDURES Chondrocytes were evaluated for type II collagen and aggrecan production They were incubated with control media or with 2 test mixtures (alone and then in combination) that have anti-inflammatory activity (avocado-soybean unsaponifiables, glucosamine, and chondroitin sulfate [ie, ASU + GLU + CS] and pentosan polysulfate and N-acetyl glucosamine [ie, PPS + NG]). Cells were then stimulated with interleukin-1β and tumor necrosis factor-α to determine prostaglandin (PG) E₂ production and nuclear factor (NF)-κB activation. RESULTS Chondrocytes proliferated in media used for propagating equine chondrocytes; they produced type II collagen and aggrecan. Cytokine stimulation induced PGE₂ production and translocation of NF-κB. Incubation with each test mixture significantly inhibited PGE₂ production. The combination of ASU + GLU + CS and PPS + NG significantly potentiated PGE₂ inhibition and disrupted NF-κB translocation, compared with effects for either mixture alone. CONCLUSIONS AND CLINICAL RELEVANCE Chondrocytes proliferated without loss of the cartilage phenotype. Responses to cytokines were significantly inhibited by the mixtures of ASU + GLU + CS and PPS + NG, which indicated that this response can be modulated. This culture technique can be used to study the functional properties of camel chondrocytes and identify agents that may potentially be used to treat and manage joint inflammation.

Hepatoprotective effects of S-adenosylmethionine and silybin on canine hepatocytes in vitro

Inflammation and oxidative stress are associated with liver injury and development of liver disease. The transcription factors nuclear factor-kappa beta (NF-κB) and nuclear factor erythroid 2-related factor 2 (Nrf2) play critical roles in modulating liver injury and damage. Activation of NF-κB induces production of pro-inflammatory molecules including prostaglandin E2 (PGE2 ), interleukin-8 (IL-8) and macrophage chemotactic protein-1 (MCP-1). Nrf2 regulates genes controlling antioxidants. Our laboratory previously showed that hepatocytes, the primary functional cell type comprising liver tissue, respond to the cytokine interleukin-1 beta (IL-1β) by increased production of PGE2 , IL-8 and MCP-1. This increase is associated with nuclear translocation of NF-κB. In this study, we evaluated whether primary canine hepatocytes pre-treated with the combination of S-adenosylmethionine (SAMe; 30 and 2000 ng/ml) and silybin (SB; 298 ng/ml), agents with known anti-inflammatory and antioxidant properties, could attenuate IL-1β-induced inflammation and oxidative stress. The SAMe and SB combination reduced cytokine-induced PGE2 , IL-8 and MCP-1 production while also inhibiting NF-κB nuclear translocation. These changes were accompanied by increased antioxidant enzyme-reduced glutathione (GSH) comparable to control levels. The study shows for the first time that the SAMe and SB combination inhibits both inflammation and oxidative stress through two separate signalling pathways.

α-Lipoic Acid Potentiates the Anti-Inflammatory Activity of Avocado/Soybean Unsaponifiables in Chondrocyte Cultures

Objective Pro-inflammatory mediators such as prostaglandin E-2 (PGE2) play major roles in the pathogenesis of osteoarthritis (OA). Although current pharmacologic treatments reduce inflammation, their prolonged use is associated with deleterious side effects prompting the search for safer and effective alternative strategies. The present study evaluated whether chondrocyte production of PGE2 can be suppressed by the combination of avocado/soybean unsaponifiables (ASU) and α-lipoic acid (LA). Design Chondrocytes from articular cartilage of equine joints were incubated for 24 hours with: (1) control media, (2) ASU, (3) LA, or (4) ASU + LA combination. Cells were activated with lipopolysaccharide (LPS), interleukin 1β (IL-1β) or hydrogen peroxide (H2O2) for 24 hours and supernatants were immunoassayed for PGE2. Nuclear factor-kappa B (NF-κB) analyses were performed by immunocytochemistry and Western blot following 1 hour of activation with IL-1β. Results LPS, IL-1β, or H2O2 significantly increased PGE2 production. ASU or LA alone suppressed PGE2 production in LPS and IL-1β activated cells. Only LA alone at 2.5 µg/mL was inhibitory in H2O2-activated chondrocytes. ASU + LA inhibited more than either agent alone in all activated cells. ASU + LA also inhibited the IL-1β induced nuclear translocation of NF-κB. Conclusions The present study provides evidence that chondrocyte PGE2 production can be inhibited by the combination of ASU + LA more effectively than either ASU or LA alone. Inhibition of PGE2 production is associated with the suppression of NF-κB translocation. The potent inhibitory effect of ASU + LA on PGE2 production could offer a potential advantage for a combination anti-inflammatory/antioxidant approach in the management of OA.

Anti-Inflammatory Effect of Carprofen Is Enhanced by Avocado/Soybean Unsaponifiables, Glucosamine and Chondroitin Sulfate Combination in Chondrocyte Microcarrier Spinner Culture

Objective Osteoarthritis is a painful, chronic joint disease affecting man and animals with no known curative therapies. Palliative nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used but they cause adverse side effects prompting the search for safer alternatives. To address this need, we evaluated the anti-inflammatory activity of avocado/soybean unsaponifiables (ASU), glucosamine (GLU), and chondroitin sulfate (CS) with or without the NSAID carprofen. Design Canine chondrocytes were propagated in microcarrier spinner culture and incubated with (1) control medium, (2) ASU (8.3 µg/mL) + GLU (11 µg/mL) + CS (20 µg/mL) combination for 24 hours; and/or carprofen (40 ng/mL). Cultures were next incubated with control medium alone or IL-1β (10 ng/mL) for another 24 hours. Production of PGE2, IL-6, IL-8, and MCP-1 (also known as CCL-2) were measured by ELISA. Results Chondrocytes proliferated in microcarrier spinner culture and produced type II collagen and aggrecan. Stimulation with IL-1β induced significant increases in PGE2, IL-6, IL-8, and MCP-1 production. The increases in production were suppressed by carprofen as well as [ASU+GLU+CS]. The combination of carprofen and [ASU+GLU+CS] reduced PGE2 production significantly more than either preparation alone. The inhibitory effect of carprofen on IL-6, IL-8, and MCP-1 production was significantly less than that of [ASU+GLU+CS], whereas the combination did not reduce the production of these molecules significantly more than [ASU+GLU+CS] alone. Conclusions The potentiating effect of [ASU+GLU+CS] on low-dose carprofen was identified in chondrocyte microcarrier spinner cultures. Our results suggest that the combination of low-dose NSAIDs like carprofen with [ASU+GLU+CS] could offer a safe, effective management for joint pain.