Anna Polotsky

AN IN VITRO SCREENING ASSAY FOR INHIBITORS OF PROINFLAMMATORY MEDIATORS IN HERBAL EXTRACTS USING HUMAN SYNOVIOCYTE CULTURES

Abstract Tumor necrosis factor–α (TNF-α), cyclooxygenase (COX)-2, and prostaglandin (PG)E-2 play a critical role in the pathophysiology of arthritis. Tumor necrosis factor–α 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 × 105 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-α, 10 ng/ml interleukin-1β, or control media alone at 5% carbon dioxide, 37° C. Cell viability, TNF-α, COX-2, PGE-2, nuclear factor κB (NF-κB), and inhibitory subunit I kappa B-alpha (IκB-α) expression were analyzed by reverse transcriptase–polymerase chain reaction, enzyme-linked immunosorbent assay, electrophoretic mobility shift assay, and Western blots. Ginger extract-HAPC (100 μg/ml) significantly inhibited the activation of TNF-α and COX-2 expression in human synoviocytes as well as suppressed production of TNF-α and PGE-2. Inhibition of TNF-α and COX-2 activation was accompanied by suppression of NF-κB and IκB-α 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.SummaryOur laboratory has shown previously that recombinant rainbow trout Ea4 (rtEa4)-peptide of pro-insulin-like growth factor-I (pro-IGF-I) exhibited antitumor activities against cancer cell lines derived from various human cancer tissues (Chen et al., 2002; Kuo and Chen, 2002). To confirm that rtEa4-peptide can exhibit the same spectrum of antitumor activities in fish tumor cells, we had developed permanent single-cell clones (RTH1B1A, RTH1B1D, RTH1B2A, and RTH1B2C) from a rainbow trout liver tumor induced by dibenzo[a,l]pyrene treatment. At 135 passages the doubling time of these single-cell clones in CO2-independent medium at 20°C was 3.9, 3.5, 3.0, and 4.5 d, respectively. Reverse transcription-polymerase chain reaction analysis showed that the expression of liver signature genes (e.g., aldolase B, glucose-6-phosphatase [G-6-Pase], phosphoenolpyruvate carboxykinase [PEPCK], hepatic nuclear factor-1 [HNF-I], IGF-I, IGF-II and growth hormone [GH] receptor-2 genes) and CYP1A1 and CYP1A3 genes was detected in these four single-cell clones. Furthermore, results of in vitro colony formation assay in a soft-agar medium showed different degrees of colony formation activities among them. These results confirmed that the single-cell clones were derived from the rainbow trout liver. Treatment of RTH1B1D with recombinant trout Ea4-peptide resulted in the induction of a dose-dependent morphological change and the suppression of colony formation in a soft-agar medium. In addition, both morphological change and reduction of colony formation were also observed in permanent transfectants of RTH1B1D cells carrying a trout Ea4-peptide gene or its human counterpart, hEb-peptide gene. These results confirm our earlier observations that trout pre-IGF-I Ea4-peptide and hEb possess activities counteracting malignant properties of cancer cells in vitro.

Beta-1 integrin expression by human nasal chondrocytes in microcarrier spinner culture.

Beta-1 integrin plays a major role in cell attachment and is believed to be involved in mediating the interactions of chondrocytes with their environment. We previously reported that articular chondrocytes propagated in microcarrier spinner culture proliferated and reexpressed their chondrocytic protein. The goal of the present study was to investigate the expression of beta-1 integrin by chondrocytes growing on the surface of microcarriers. Nasal chondrocytes (4 x 10(3)/cm(2)) were seeded on microcarriers and incubated at 37 degrees C, 5% CO(2), 60 rpm. Expression of chondrocyte markers and beta-1 integrin was determined using reverse transcriptase-polymerase chain reaction and immunocytochemical analyses. De novo synthesis of sulfate-containing proteoglycans was studied using 35SO(4) incorporation techniques. Like articular chondrocytes propagated in microcarrier spinner culture, nasal chondrocytes expressed high levels of collagen type II mRNA, whereas collagen type I mRNA levels were low. Aggrecan mRNA was detectable and levels of de novo 35SO(4) incorporation were high. Chondrocytes immunostained intensely for collagen type II and keratan sulfate but did not stain for collagen type I. beta-1 integrin mRNA levels were high, and the protein was immunolocalized to regions of cell-to-cell or cell-to-microcarrier contact. The fact the chondrocytes expressed high levels of beta-1 integrin raises the possibility that this integrin molecule has a role in the maintenance of the chondrocytic phenotype.

COLLAGEN MICROCARRIER SPINNER CULTURE PROMOTES OSTEOBLAST PROLIFERATION AND SYNTHESIS OF MATRIX PROTEINS

SummaryIn 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 produced 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.

Propagation of human nasal chondrocytes in microcarrier spinner culture.

Objective The aim of this study was to test the effectiveness of nasal septal chondrocytes, propagated in microcarrier spinner culture, as an alternative tissue source of chondrocytic cells for cartilage grafts for head and neck surgery and for articular cartilage repair. Methods We harvested chondrocytes from 159 patients, ranging in age from 15 to 80 years and undergoing repair of a deviated nasal septum, and propagated the cells in a microcarrier spinner culture system. The nasal chondrocytes proliferated and produced extracellular matrix components similar to that produced by articular chondrocytes. Results In microcarrier spinner culture on collagen beads, chondrocyte numbers increased up to 14-fold in 2 weeks. After a month, the microcarriers seeded with nasal chondrocytes began to aggregate, producing a dense cartilage-like material. The newly synthesized extracellular matrix was rich in high molecular weight proteoglycans, and the chondrocytes expressed type II collagen and aggrecan but not type I collagen. Conclusion These studies support the feasibility of engineering cartilage tissue using chondrocytes harvested from the nasal septum. Injectable and solid formulations based on this technology are being evaluated for applications in craniomaxillofacial reconstructive surgery and for plastic and orthopedic surgery practices.

Cyclic strain stimulates proliferative capacity, α2 and α5 integrin, gene marker expression by human articular chondrocytes propagated on flexible silicone membranes

SummaryChondrocytes 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 α2 and α5 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.

Fluoride-Substituted Apatites Support Proliferation and Expression of Human Osteoblast Phenotype In Vitro

This study aimed to test the hypothesis that fluoride (F) ions rele as d from F-substituted apatite (FAp) modulate osteoblast behavior in vitro. FAp samples of varying F concentrations (0.01, 0.08, 2.01 and 3.34 wt% F) were used in this study. Human osteoblasts (1x10 /well) were incubated in the presence or absence of Fap (5mg/well) for four days at 37C, 5% CO2. Viability, proliferative capacity by radiolabeled thymidine uptake and RNA were d t rmined. Results showed the following: viability and proliferative capacity of cells expos ed to FAp particle suspension were similar to control cells; collagen Type 1 expression was compara ble in all groups whereas alkaline phosphatase expression was variable; and osteocalcin expression was e nhanced in osteoblasts cells exposed to FAp with F concentrations 0.08 wt% and higher. These results de monstrated that F ions released from F-substituted apatites support proliferative capacit y of human osteoblast cells and enhance osteocalcin expression. These findings suggest that fluoride m ay play a role in bone formation and therefore F-releasing bone graft materials may provide added advanta ge.

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

SummaryThe 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° 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.

INDUCTION OF OSTEOBLAST AGGREGATION, DETACHMENT, AND ALTERED INTEGRIN EXPRESSION BY BEAR SERUM

SummaryAnimal 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.