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-1ß 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.

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-1ß (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-1ß 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.

Avocado/Soybean Unsaponifiables, Glucosamine and Chondroitin Sulfate Combination Inhibits Proinflammatory COX-2 Expression and Prostaglandin E2 Production in Tendon-Derived Cells.

Tendinopathy, a common disorder in man and horses, is characterized by pain, dysfunction, and tendon degeneration. Inflammation plays a key role in the pathogenesis of tendinopathy. Tendon cells produce proinflammatory molecules that induce pain and tissue deterioration. Currently used nonsteroidal anti-inflammatory drugs are palliative but have been associated with adverse side effects prompting the search for safe, alternative compounds. This study determined whether tendon-derived cells' expression of proinflammatory cyclooxygenase (COX)-2 and production of prostaglandin E2 (PGE2) could be attenuated by the combination of avocado/soybean unsaponifiables (ASU), glucosamine (GLU), and chondroitin sulfate (CS). ASU, GLU, and CS have been used in the management of osteoarthritis-associated joint inflammation. Tenocytes in monolayer and microcarrier spinner cultures were incubated with media alone, or with the combination of ASU (8.3 µg/mL), GLU (11 µg/mL), and CS (20 µg/mL). Cultures were next incubated with media alone, or stimulated with interleukin-1ß (IL-1ß; 10 ng/mL) for 1 h to measure COX-2 gene expression, or for 24 h to measure PGE2 production, respectively. Tenocyte phenotype was analyzed by phase-contrast microscopy, immunocytochemistry, and Western blotting. Tendon-derived cells proliferated and produced extracellular matrix component type I collagen in monolayer and microcarrier spinner cultures. IL-1ß-induced COX-2 gene expression and PGE2 production were significantly reduced by the combination of (ASU+GLU+CS). The suppression of IL-1ß-induced inflammatory response suggests that (ASU+GLU+CS) may help attenuate deleterious inflammation in tendons.

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 1ß (IL-1ß) 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-1ß. Results LPS, IL-1ß, or H2O2 significantly increased PGE2 production. ASU or LA alone suppressed PGE2 production in LPS and IL-1ß 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-1ß 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.

Microcarriers in the engineering of cartilage and bone.

A major problem in tissue engineering is the availability of a sufficient number of cells with the appropriate phenotype for delivery to damaged or diseased cartilage and bone; the challenge is to amplify cell numbers and maintain the appropriate phenotype for tissue repair and restoration of function. The microcarrier bioreactor culture system offers an attractive method for cell amplification and enhancement of phenotype expression. Besides serving as substrates for the propagation of anchorage-dependent cells, microcarriers can also be used to deliver the expanded undifferentiated or differentiated cells to the site of the defect. The present article provides an overview of the microcarrier culture system, its utility as an in vitro research tool and its potential applications in tissue engineering, particularly in the repair of cartilage and bone.

Nickel and vanadium metal ions induce apoptosis of T-lymphocyte Jurkat cells.

Metal alloys are used as prosthetic components in the orthopaedic and dental field. However, there is growing concern over the reported leaching of metal ions from implants. Ions released from metals have been thought to be associated with local immune dysfunction, inflammation, and tissue cell death. The objective of our study was to investigate whether nickel(II) and vanadium(V), present at a smaller percentage in most alloys, are cytotoxic to T-lymphocyte cell models. Jurkat T cells possess characteristics similar to human T-lymphocytes and proliferate at a faster rate. Jurkat T cells were incubated with control media alone or with concentrations of 1, 10, and 100 microg/mL of Ni(II) or V(V) for 24 h. Both types of metal ions reduced cell viability and proliferation in a dose-dependent manner. Ni(II) at 10 microg/mL and V(V) at 100 microg/mL activated Caspase-3 expression. Hoechst 33258 staining and transmission electron microscopy revealed chromatin condensation, as well as nuclear blebbing and fragmentation. Induction of DNA fragmentation by Ni(II) at 100 microg/mL was also indicated by agarose electrophoresis. Our observations indicate that Ni and V ions kill T cells via apoptotic and nonapoptotic pathways.

Immolina, a high-molecular-weight polysaccharide fraction of Spirulina, enhances chemokine expression in human monocytic THP-1 cells.

INTRODUCTION: Spirulina (Spirulina platensis) is a dietary supplement valued for its immune-enhancing properties. We previously reported that the immunostimulatory effect of spirulina can be traced to a high-molecular- weight polysaccharide fraction. This fraction, labeled Immolina, activates nuclear factor kappa-B in human monocytic THP-1 cells and increases expression of proinflammatory cytokines. OBJECTIVE: To characterize further the immunostimulatory effects of Immolina on THP-1 cells, we evaluated its effect on genes encoding the chemokines interleukin (IL)-8, MCP-1, MIP-1alpha, MIP-1beta, IP-10, the cytokines tumor necrosis factor (TNF)-alpha, IL-1beta, and the enzyme cyclo-oxygenase-2 (COX-2). METHODS: THP-1 cells were exposed to concentrations of Immolina ranging from 1 ng/mL to 100 microg/mL and changes in gene expression were assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). For comparison, THP-1 cells were activated with 1 ng/mL of TNF-alpha, 10 ng/mL of IL-1beta, or 10 ng/mL of lipopolysaccharide using the same assay conditions. To assess the response of THP-1 cells to Immolina at the protein level, we probed culture supernatants using a cytokine array immunoblot assay. RESULTS: RT-PCR analysis revealed that Immolina dose-dependently increased the expression of all 5 chemokines tested as well as the expression of TNF-alpha, IL-1beta, and COX-2. The cytokine array immunoblot assay revealed an increase in the chemokines IL-8 and MIP-1beta. Thymidine uptake experiments verified that Immolina did not affect the viability and growth rate of THP-1 cells. CONCLUSIONS: The results of the experiments demonstrate that Immolina activates THP-1 cells in a manner that is consistent with the recruitment of diverse populations of leukocytes in response to inflammatory and infectious signals.

Ginger--an herbal medicinal product with broad anti-inflammatory actions.

The anti-inflammatory properties of ginger have been known and valued for centuries. During the past 25 years, many laboratories have provided scientific support for the long-held belief that ginger contains constituents with antiinflammatory properties. The original discovery of ginger's inhibitory effects on prostaglandin biosynthesis in the early 1970s has been repeatedly confirmed. This discovery identified ginger as an herbal medicinal product that shares pharmacological properties with non-steroidal anti-inflammatory drugs. Ginger suppresses prostaglandin synthesis through inhibition of cyclooxygenase-1 and cyclooxygenase-2. An important extension of this early work was the observation that ginger also suppresses leukotriene biosynthesis by inhibiting 5-lipoxygenase. This pharmacological property distinguishes ginger from nonsteroidal anti-inflammatory drugs. This discovery preceded the observation that dual inhibitors of cyclooxygenase and 5-lipoxygenase may have a better therapeutic profile and have fewer side effects than non-steroidal anti-inflammatory drugs. The characterization of the pharmacological properties of ginger entered a new phase with the discovery that a ginger extract (EV.EXT.77) derived from Zingiber officinale (family Zingiberaceae) and Alpina galanga (family Zingiberaceae) inhibits the induction of several genes involved in the inflammatory response. These include genes encoding cytokines, chemokines, and the inducible enzyme cyclooxygenase-2. This discovery provided the first evidence that ginger modulates biochemical pathways activated in chronic inflammation. Identification of the molecular targets of individual ginger constituents provides an opportunity to optimize and standardize ginger products with respect to their effects on specific biomarkers of inflammation. Such preparations will be useful for studies in experimental animals and humans.

Ginger extract components suppress induction of chemokine expression in human synoviocytes.

INTRODUCTION: Ginger has a long history of medicinal use, particularly as an anti-inflammatory agent for a wide variety of diseases such as arthritis. Suppression of inflammation in arthritis is attributed to suppression of proinflammatory cytokines and chemokines produced by synoviocytes, chondrocytes, and leukocytes. OBJECTIVE: This study aimed to elucidate the effect of a combination ginger extract and its individual components on chemokine expression in human synoviocytes. METHODS: Human synoviocytes were incubated with 100 microg/mL combination ginger extract (GE) of Alpinia galanga (AG) and Zingiber officinale (ZO); AG extract alone; ZO extract alone; or control media, for 1 hour at 37 degrees C, 5% CO2. Cells were next activated with 1 ng/mL of tumor necrosis factor alpha (TNF-alpha) for 1 hour to determine macrophage chemotactic factor (MCP-1) and interferon-gamma activated protein (IP-10) mRNA levels using reverse transcriptase polymerase chain reaction (RT-PCR). Secreted MCP-1 and IP-10 were quantified by enzyme-linked immunosorbent assay (ELISA) following a 24 hour incubation period. RESULTS: The GE combination was consistently more effective in decreasing chemokine mRNA and chemokine secreted protein levels than its individual components ZO or AG. In comparison, ZO was more effective than AG in suppressing chemokine expression. CONCLUSION: The present study demonstrates that GE inhibits chemokine expression, and that the combination of ZO and AG components acts synergistically. This ginger formulation may be useful for suppressing inflammation due to arthritis.

Thermally reversible polymer gel for chondrocyte culture.

We have evaluated a biomaterial to serve as a scaffold for the propagation and amplification of chondrocytes that promotes the original cellular phenotype of these cells. The goal of the present study was to investigate the use of thermally reversible polymer gels poly(NiPAAm-co-AAc), as a biocompatible supporting scaffold for the propagation of chondrocytic cells. The polymer gels at temperatures above its lower critical solution temperature whereas liquefying at temperatures below its lower critical solution temperature of 34.5 degrees C. Hence, the polymer, in its gelled form, has the ability to hold cells in situ, forming a matrix similar to the natural cellular environment or the extracellular matrix that comprises cartilage. We tested the hypothesis that the polymer gel promotes cell viability and function. Human osteoblast-like cells, nasal chondrocytes, and articular chondrocytes (1 x 10(5)/150 microL) were resuspended in enriched Dulbecco's minimal essential media and were plated onto control (without gel) and gel containing 24-well plates. The plates were reincubated at 37 degrees C, 5% CO(2) for the time point of interest. Additional media was added to the plates and exchanged as needed. After cell culture, cells were retrieved, enumerated, and cell viability was determined. Other aliquots of the cells were stained for morphological analysis whereas expression of chondrocyte markers including collagen type II and aggrecan were determined using reverse transcriptase-polymerase chain reaction. The polymer gel was not cytotoxic because the cell number retrieved from three-dimensional culture gel was found to be one to two times higher than that retrieved from monolayer culture. Chondrocytes propagated in the thermo-reversible polymers expressed enhanced or maintained expression of collagen type II and aggrecan. Collagen type I expression was decreased or unaltered. The N-isopropylacrylamide and acrylic acid copolymer gel has potential use as a cell culture substrate and as a cell delivery vehicle.

Evaluation of thermoreversible polymers containing fibroblast growth factor 9 (FGF-9) for chondrocyte culture.

We previously evaluated a thermoreversible polymer gel composed of N-isopropylacrylamide and acrylic acid as a cell culture substrate and cell-delivery vehicle. The copolymer promoted phenotype expression and amplification of chondrocytes. In this study, we determined whether addition of fibroblast growth factor 9 (FGF-9), which is mitogenic for chondrocytes, would further enhance cell proliferation and phenotype expression in the polymer. We tested the hypothesis that the thermoreversible polymer containing FGF-9 would promote increased chondrocyte proliferation and phenotype expression. Articular chondrocytes (1 x 10(5)/150 microL) were plated onto control (without gel) and gel containing 24-well plates. The gels were prepared in media alone or in media containing heparin (100 microg/mL) and FGF-9 (5 microg/mL). The cultures were incubated at 37 degrees C in 5% CO(2) for 3 days. Cells remained viable in the thermoreversible polymer in the presence or absence of FGF-9. Addition of FGF-9 to the copolymer did not induce proliferation and the cell numbers did not increase. Reverse transcription polymerase chain reaction (RT-PCR)-determined expression of chondrocyte markers collagen type II and aggrecan. FGF-9 did not enhance chondrocyte proliferation nor alter the phenotype after 3 days in culture. These findings suggest the poly(NiPA-co-AAc) gel alone may provide the optimal 3D environment for propagation of chondrocytes.

The effect of silica-containing calcium-phosphate particles on human osteoblasts in vitro.

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.

Cyclic strain stimulates proliferative capacity, alpha2 and alpha5 integrin, gene marker expression by human articular chondrocytes propagated on flexible silicone membranes.

Chondrocytes comprise less than 10% of cartilage tissue but are responsible for sensing and responding to mechanical stimuli imposed on the joint. However, the effect of mechanical signals at the cellular level is not yet fully defined. The purpose of this study was to test the hypothesis that mechanical stimulation in the form of cyclic strain modulates proliferative capacity and integrin expression of chondrocytes from osteoarthritic knee joints. Chondrocytes isolated from articular cartilage during total knee arthroplasty were propagated on flexible silicone membranes. The cells were subjected to cyclic strain for 24 h using a computer-controlled vacuum device, with replicate samples maintained under static conditions. Our results demonstrated increase in proliferative capacity of the cells subjected to cyclic strain compared with cells maintained under static conditions. The flexed cells also exhibited upregulation of the chondrocytic gene markers type II collagen and aggrecan. In addition, cyclic strain resulted in increased expression of the alpha2 and alpha5 integrin subunits, as well as an increased expression of vimentin. There was also intracellular reconfiguration of the enzyme protein kinase C. Our findings suggest that these molecules may play a role in the signal transduction pathway, eliciting cellular response to mechanical stimulation.

Induction of osteoblast aggregation, detachment, and altered integrin expression by bear serum.

Animal models have long been used to elucidate the mechanisms responsible for osteoporosis in humans. The American black bear, an animal that does not experience extensive bone loss normally associated with long-term immobilization (when hibernating), may provide an insight into the nature of the pathogenesis of the disease. Circulating growth and differentiation factors present in the serum may facilitate continued proliferation of bone-forming cells. The aim of our study was to determine the effects of bear serum on human osteoblasts when cultured for extended periods of time. Unexpectedly, exposure to the bear serum in vitro led to the detachment of osteoblasts from the surface of the culture plate after 3 d of incubation. The osteoblasts pulled off the polystyrene surface in sheets and aggregated into floating conglomerations of viable cells. In contrast, osteoblasts cultured in fetal calf serum maintained adherence to the surface of the culture plate. Detachment of osteoblasts propagated in bear serum was time dependent and was associated with an increased expression of integrins compared with osteoblasts propagated in fetal calf serum, as indicated by reverse transcriptase-polymerase chain reaction and immunostaining.

Collagen microcarrier spinner culture promotes osteoblast proliferation and synthesis of matrix proteins.

In vitro propagation of osteoblasts in three-dimensional culture has been explored as a means of cell line expansion and tissue engineering purposes. Studies investigating optimal culture conditions are being conducted to produce bone-like material. This study demonstrates the use of collagen microcarrier beads as a substrate for three-dimensional cell culture. We have earlier reported that microcarriers consisting of cross-linked type I collagen support chondrocyte proliferation and synthesis of extracellular matrix. In this study, we investigated the use of collagen microcarriers to propagate human trabecular bone-derived osteoblasts. Aggregation of cell-seeded microcarriers and production of extracellular matrix-like material were observed after 5 d in culture. Expression of extracellular matrix proteins osteocalcin, osteopontin, and type I collagen was confirmed by messenger ribonucleic acid analysis, radioimmunoassay, and Western blot analysis. The efficient recovery of viable cells was achieved by collagenase digestion of the cell-seeded microcarriers. The collagen microcarrier spinner culture system provides an efficient method to amplify large numbers of healthy functional cells that can be subsequently used for further in vitro or transplantation studies.

An in vitro screening assay for inhibitors of proinflammatory mediators in herbal extracts using human synoviocyte cultures.

Tumor necrosis factor-alpha (TNF-alpha), cyclooxygenase (COX)-2, and prostaglandin (PG)E-2 play a critical role in the pathophysiology of arthritis. Tumor necrosis factor-alpha mediates induction of other cytokines, COX-2, PGs, and metalloproteinases, which leads to cartilage degradation. We developed an in vitro human synoviocyte assay system for screening inhibitors of proinflammatory mediators in herbal extracts. Synoviocytes (5 x 10(5) cells/well) obtained during primary knee replacement from osteoarthritic patients were incubated with: control media alone or ginger extract (hydroxy-methoxy-phenyl compounds [HAPC]: EV.EXT 77), 1 h before activation with 1 ng/ml TNF-alpha, 10 ng/ml interleukin-1beta, or control media alone at 5% carbon dioxide, 37 degrees C. Cell viability, TNF-alpha, COX-2, PGE-2, nuclear factor kappaB (NF-kappaB), and inhibitory subunit I kappa B-alpha (IkappaB-alpha) expression were analyzed by reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, electrophoretic mobility shift assay, and Western blots. Ginger extract-HAPC (100 microg/ml) significantly inhibited the activation of TNF-alpha and COX-2 expression in human synoviocytes as well as suppressed production of TNF-alpha and PGE-2. Inhibition of TNF-alpha and COX-2 activation was accompanied by suppression of NF-kappaB and IkappaB-alpha induction. Using our in vitro assay, we discovered that the ginger extract blocks activation of proinflammatory mediators and its transcriptional regulator suggesting its mode of action. These observations indicate that ginger extract-HAPC offers a complementary and alternative approach to modulate the inflammatory process involved in arthritis.

Enhancement of osteoblast proliferative capacity by growth factor-like molecules in bear serum.

The use of animal serum in cell culture is vital for providing the nutrient factors required to promote proliferation and function. Fetal calf serum has become the preferred choice because of its abundance, reasonable cost, and ability to sustain human cells in vitro. Although a wide variety of serum sources have been tested and used, little is known about the ability of serum obtained from the American black bear (Ursus americanus) to support human cell growth in culture. The American black bear, an animal comparable in size to humans, is unique in that it hibernates for mo at a time but does not experience extensive bone loss normally associated with extended immobility. The aim of this study was to analyze the effect of bear serum on human osteoblast cultures. We discovered that three of the eight bear serum samples induced significantly higher proliferation rates in osteoblasts than did fetal calf serum over a 24-h period. Osteoblasts incubated in bear serum displayed higher messenger ribonucleic acid levels for phenotype markers osteocalcin and type I collagen than did those incubated in fetal calf serum. The mitogenic activity of the bear serum was reduced when heated at 56 degrees C for 30 min before use in culture. The molecular weight of the mitogenic factors was found to be primarily greater than 50 kDa. The present work demonstrates the capability of serum from American black bears to support human osteoblast proliferation in vitro.

Cartilage debris increases the expression of chondrodestructive tumor necrosis factor-alpha by articular chondrocytes.

PURPOSE: To determine if cartilage particles increased the expression of TNF-alpha by articular chondrocytes. TYPE OF STUDY: In vitro experiment. METHODS: Articular chondrocytes were obtained from patients undergoing primary total knee arthroplasty for osteoarthritis (n = 3) and from patients undergoing below-knee amputation for peripheral vascular disease (n = 3). Chondrocytes were then incubated with and without cartilage particles at a concentration of 5 microg/10(5) cells for 24 hours. TNF-alpha levels were then determined using reverse transcription polymerase chain reaction. RESULTS: Both normal and osteoarthritic chondrocytes had low baseline expression of TNF-alpha under standard cell culture conditions. Expression was markedly increased in response to incubation with cartilage particles, and was statistically significant. CONCLUSIONS: Cartilage debris in the traumatized and osteoarthritic joint may increase the concentration of TNF-alpha in the joint, contributing to joint symptoms and cartilage destruction. Arthroscopic debridement and lavage may improve symptoms by washing these harmful components from the joint. LEVEL OF EVIDENCE: Level IV.

Ginger extract inhibits beta-amyloid peptide-induced cytokine and chemokine expression in cultured THP-1 monocytes.

INTRODUCTION: Neuritic plaques, a neuropathologic hallmark of Alzheimer's disease, are extracellular deposits of beta-amyloid peptides (Abeta). In the central nervous system neuritic plaques are surrounded by activated microglial cells expressing proinflammatory cytokines, chemokines, and neurotoxic mediators. Long-term activation of microglial cells is suspected to contribute to the neuron loss in Alzheimer's disease. OBJECTIVE: This study was conducted to determine whether a ginger (Zingiber officinale and Alpinia galanga) extract (GE) can dampen the activation of THP-1 cells by lipopolysaccharide, proinflammatory cytokines, and fibrillar amyloid peptide Abeta(1-42), a major component of neuritic plaques. METHODS: THP-1 cells, a human monocytic cell line with properties similar to human microglial cells, were incubated with GE or control medium alone for 1 hour, and then with reincubated lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) or fibrillar Abeta(1-42) for an additional hour. The extent of THP-1 cell activation was determined by measuring mRNA levels of TNF-alpha and IL-1beta, cyclooxygenase-2 (COX-2), macrophage inflammatory protein 1alpha (MIP-1alpha), monocyte chemoattractant protein-1 (MCP-1), and interferon-gamma inducible protein 10 (IP-10). RESULTS: The results document that the GE used in this study inhibits LPS, cytokine, and amyloid Abeta peptide-induced expression of the proinflammatory genes TNF-alpha, IL-1beta, COX-2, MIP-alpha, MCP-1, and IP-10. The data provide experimental evidence that ginger can inhibit the activation of human monocytic THP-1 cells by different proinflammatory stimuli and reduce the expression of a wide range of inflammation-related genes in these microglial-like cells. CONCLUSIONS: The findings suggest that GE may be useful in delaying the onset and the progression of neurodegenerative disorders involving chronically activated microglial cells in the central nervous system.

Expression of pro-inflammatory mediators is inhibited by an avocado/soybean unsaponifiables and epigallocatechin gallate combination.

BACKGROUND: Osteoarthritis (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. FINDINGS: Articular 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-1ß, 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-1ß, 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-1ß, TNF-α, IL-6, COX-2, and IL-8 in cytokine-activated chondrocytes. Western blot and immunostaining confirmed NF-κB translocation inhibition. CONCLUSIONS: We demonstrate that ASU + EGCG inhibits cytokine-induced gene expression of IL-1ß, 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.