The present study demonstrates that the anti-inflammatory activity of ASU and EGCG is potentiated when used in combination. This combination may offer an attractive supplement or alternative to non-steroidal anti-inflammatory drugs (NSAIDs) in the management of osteoarthritis.

There is an ongoing need for more effective and less costly bone substitutes. It has previously been proposed that silica-containing bioactive glass would be more effective as a bone repair material because of its physiochemical properties. Three newly synthesized silica-containing bioactive glass formulations, HA-31 (25%), HA-11 (50%), and HA-13 (75%), were tested as biocompatible substrates for the continued proliferation and phenotype expression of human bone cells in vitro. Two currently available bioactive glasses (BioGlass(R), Hydroxyapatite) served as comparisons. The biocompatibility of these bioglasses, as well as their osteoconductive properties, was assessed by employing primary cultures of human osteoblasts and human synoviocytes for 4 days. The results obtained demonstrated that the three new bioglasses enhanced the proliferative response of osteoblasts compared with osteoblasts cultured alone. Reverse Transcription Polymerase Chain Reaction (RT-PCR) analysis indicated that osteoblasts retained their phenotypic expression by continued expression of collagen type I and alkaline phosphatase. The newly synthesized preparations of silica-containing bioactive glass did not induce stimulation of proinflammatory markers iNOS and IL-1beta in synoviocytes. In conclusion, the newly synthesized silica-containing bioactive glasses are biocompatible substrate for bone-forming osteoblasts. However, the formulations tested did not show significant advantage over the currently available bioactive glasses in vitro.

BackgroundOsteoarthritis (OA) is characterized by inflammation, joint immobility, and pain. Non-pharmacologic agents modulating pro-inflammatory mediator expression offer considerable promise as safe and effective treatments for OA. We previously determined the anti-inflammatory effect of an avocado/soybean unsaponifiables (ASU) and epigallocatechin gallate (EGCG) combination on prostaglandin E2 (PGE2) production and nuclear factor-kappa B (NF-κB) translocation. The aim of this study was to evaluate the effects of ASU + EGCG on pro-inflammatory gene expression.FindingsArticular chondrocytes from carpal joints of mature horses were pre-incubated for 24 hours with control media alone or ASU (8.3 μg/mL) + EGCG (40 ng/mL), followed by one hour activation with interleukin-1 beta (IL-1β, 10 ng/mL) and tumor necrosis factor-alpha (TNF-α, 1 ng/mL). Total cellular RNA was isolated and real-time PCR performed to measure IL-1β, TNF-α, interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), and interleukin-8 (IL-8) gene expression. Intracellular localization of NF-κB was analyzed by immunohistochemistry and Western blot. Pre-treatment with ASU + EGCG significantly (P < 0.001) decreased gene expression of IL-1β, TNF-α, IL-6, COX-2, and IL-8 in cytokine-activated chondrocytes. Western blot and immunostaining confirmed NF-κB translocation inhibition.ConclusionsWe demonstrate that ASU + EGCG inhibits cytokine-induced gene expression of IL-1β, TNF-α, IL-6, COX-2, and IL-8 through modulation of NF-κB. Our results indicate that the activity of ASU + EGCG affects a wide array of inflammatory molecules in addition to decreasing PGE2 synthesis in activated chondrocytes. The responsiveness of chondrocytes to this combination supports its potential utility for the inhibition of joint inflammation.

Objective Pro-inflammatory mediators such as prostaglandin E-2 (PGE) 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 PGE 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 (HO) for 24 hours and supernatants were immunoassayed for PGE. 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 HO significantly increased PGE production. ASU or LA alone suppressed PGE production in LPS and IL-1β activated cells. Only LA alone at 2.5 µg/mL was inhibitory in HO-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 PGE production can be inhibited by the combination of ASU + LA more effectively than either ASU or LA alone. Inhibition of PGE production is associated with the suppression of NF-κB translocation. The potent inhibitory effect of ASU + LA on PGE production could offer a potential advantage for a combination anti-inflammatory/antioxidant approach in the management of OA.

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.

iii ACKNOWLEDGEMENTS

Osteoarthritis (OA) is a chronic, painful disease affecting articulating joints in man and animals. It is characterized by cartilage breakdown, bone remodeling, osteophyte formation and joint inflammation. Currently used non-steroidal anti-inflammatory drugs for the management of OA are known to have deleterious side effects. To address the need for alternatives, we evaluated the anti-inflammatory effects of a combination of avocado/soybean unsaponifiables (ASU), glucosamine (GLU) and chondroitin sulfate (CS) by measuring chemokine MCP-1 (monocyte chemoattractant protein 1, CCL2) and prostaglandin E-2 (PGE 2 ) in stimulated chondrocytes. As the only cellular constituents of cartilage, chondrocytes are the source of pro-inflammatory mediators that play critical roles in the pathogenesis of OA. Chondrocytes were incubated: with: 1) control media, 2) [ASU + GLU + CS] combination, or 3) Phenylbutazone (PBZ) for 24 hours. Cells were next stimulated with IL-1β or LPS for another 24 hrs. MCP-1 and PGE 2 from supernatants were quantitated by immunoassay. Another set of chondrocytes seeded in chamber slides were stimulated with IL-1β for 1 hour and then immunostained for NF-κB. Chondrocytes stimulated with IL-1β or LPS significantly increased MCP-1 and PGE 2 production which were significantly reduced after treatment with [ASU + GLU + CS]. In contrast, PBZ significantly reduced PGE 2 but not MCP-1 production. IL-1β stimulation induced nuclear translocation of NF-κB, which The present study provides evidence that the production of MCP-1 by chondrocytes can be inhibited by the combination of [ASU + GLU + CS] but not by PBZ. In contrast, PGE 2 production was inhibited by either treatment suggesting that the production of MCP-1 and PGE 2 could be independently regulated. The finding of distinct effects of [ASU + GLU + CS] on MCP-1 and PGE 2 synthesis supports a scientific rationale for a multimodal treatment approach in the management of OA.

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