Carmelita G. Frondoza

Chitosan supports the expression of extracellular matrix proteins in human osteoblasts and chondrocytes

The search for biocompatible materials that can support the growth and phenotypic expression of osteoblasts and chondrocytes is a major challenge in the application of tissue engineering techniques for the repair of bone and cartilage defects. Chitosan, a copolymer of glucosamine and N-acetylglucosamine, may provide an answer to this search. Chitosan is the deacetylated product of chitin, a ubiquitous biopolymer found in the exoskeleton of insects and marine invertebrates. Little is known about the utility of chitosan in propagating human osteoblasts and chondrocytes. In this study, we test the hypothesis that chitosan promotes the survival and function of osteoblasts and chondrocytes. Chitosan (4%, w/v in 2% HAc) was coated onto plastic coverslips that had been fitted into 24-well plates. Human osteoblasts and articular chondrocytes were seeded on either uncoated or chitosan-coated coverslips at 1 x 10(5)/cells per well. Cultures were incubated at 37 degrees C, 5% CO(2) for a period of 7 days. Cell viability was assessed at that time using a fluorescent molecular probe. The phenotypic expression of osteoblasts and chondrocytes was analyzed by reverse transcriptase-polymerase chain reaction and immunocytochemistry. Osteoblasts and chondrocytes appeared spherical and refractile on chitosan-coated coverslips. In contrast, greater than 90% of cells on plastic coverslips were elongated and spindle shaped after 7 days of culture. Similar to cells propagated on uncoated control wells, greater than 90% of human osteoblasts and chondrocytes propagated on chitosan remained viable. Human osteoblasts propagated on chitosan films continued to express collagen type I whereas chondrocytes expressed collagen type II and aggrecan, as shown by reverse transcriptase-polymerase chain reaction analysis and immunostaining. The present in vitro work demonstrates the biocompatibility of chitosan as a substrate for the growth and continued function of human osteoblasts and chondrocytes. Chitosan may have potential use as a tissue engineering tool for the repair of osseous and chondral defects.

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.

Regulation of osteocalcin secretion by human primary bone cells and by the human osteosarcoma cell line MG-63

We present evidence that the regulation of osteocalcin secretion by PTH and PGE2 in normal human bone cells can be produced in the human osteoblast-like cell line MG-63. Both cell cultures showed time- and dose-dependent stimulation of osteocalcin secretion in response to 1,25(OH)2D3. Bovine parathyroid hormone (PTH) amino acid fragment 1-34 (40 nM) and prostaglandin E2 (PGE2, 5 nM) significantly inhibited 1,25(OH)2D3-induced osteocalcin secretion by these cells. The inhibition reached 20 and 36%, respectively. In contrast, PTH 3-34 had no effect on osteocalcin secretion. Both cell cultures produced cAMP in response to PTH. Dexamethasone (Dex) (100 nM) potentiated PTH-induced (40 nM) cAMP synthesis in subconfluent MG-63 cells (1.5-fold increase, P less than 0.05). This treatment with Dex resulted in a greater inhibition of 1,25(OH)2D3-induced osteocalcin secretion (-30%, P less than 0.005) by PTH in MG-63 cells as compared to cells exposed to PTH and 1,25(OH)2D3 alone. Pretreatment of subconfluent MG-63 cells with Dex (100 nM) for 48 h also increased 1,25(OH)2D3-induced osteocalcin secretion by 40% (P less than 0.025). In contrast, treatments of confluent MG-63 cells with Dex inhibited osteocalcin secretion regardless of the 1,25(OH)2D3 doses used. Forskolin (10(-7)-10(-5) M) and dibutyryl cAMP (10(-6)-(10(-3) M) both reproduced the effects observed with PTH and PGE2 in the two cell cultures. Forskolins action was time-dependent: addition of forskolin (10(-6) M) 12 h after 1,25(OH)2D3 (50 nM) resulted in a progressively weaker inhibition of osteocalcin secretion. Increasing the extracellular calcium concentration of the incubation media resulted in a dose-dependent increase in osteocalcin secretion (P less than 0.01). These results indicate that PTH and PGE2 inhibit osteocalcin secretion by a mechanism involving cAMP production. In contrast, an increase in extracellular calcium stimulated osteocalcin release. Thus the human osteosarcoma cell line MG-63 is a useful osteoblast-like cell model to study the regulation of osteocalcin secretion. Furthermore, a factor (or factors) between hormone-receptor coupling and gene induction can regulate the expression of the osteocalcin gene or affect pre- or posttranslational mechanisms implicated in osteocalcin synthesis and secretion.

Effect of prosthetic titanium wear debris on mitogen-induced monocyte and lymphoid activation.

Wear debris generated by joint implant components has been reported to activate inflammatory and immune cells. Particulate debris derived from prosthetic material induces monocytes/macrophages, lymphocytes, synoviocytes, and fibroblasts to secrete cellular products, such as cytokines, which mediate inflammation. It has been speculated that degradation products impair the ability of inflammatory and immune cells to mount a protective response against noxious agents and infectious organisms by interfering with cell activation. Recent in vitro studies suggest that soluble metal ions inhibit T and B cell activation, but it is not known whether insoluble metal particles generated by prosthetic wear in tissue have the same effect. The purpose of the present study was to determine whether titanium wear debris retrieved from periprosthetic tissues surrounding a failed knee prosthesis suppresses activation of human monocytic and lymphoid cells. Peripheral blood monocytes and lymphocytes were incubated with the nonspecific activator pokeweed mitogen (PWM) in the presence or absence of titanium particles. Cell proliferative capacity and production of interleukins IL-1beta and IL-2 were determined as measures of activation. Titanium wear debris induced monocyte secretion of IL-1beta at levels comparable to those induced by PWM alone. In combination with PWM, titanium wear debris stimulated monocytes to secrete higher concentrations of IL-1beta than is stimulated by titanium itself or by PWM alone. Titanium wear debris did not activate lymphocytes, as indicated by marginal changes in DNA synthesis and IL-2 secretion, nor did it suppress the PWM-induced stimulation of DNA synthesis and IL-2 secretion. Our study suggests that nonspecific mitogen activators in spite of exposure to titanium wear debris can stimulate monocytic and lymphoid cells.

Glass peek composite promotes proliferation and osteocalcin production of human osteoblastic cells

An isoelastic intramedullary implant has been developed using a composite of polyetheretherketone and 10% random, chopped E-glass fibers (GPEEK). The effect of this novel material on human bone cells has not been defined. The objective of this study was to test whether GPEEK supported the proliferation of the human bone cell line MG63, which exhibits osteoblastlike characteristics. Cells (1 x 10(5)/mL) were propagated on GPEEK discs with three different surface roughnesses (3, 6, and 9 microns) and on polystyrene plates, for comparison. The reaction of MG63 osteoblastlike cells to the GPEEK polymer composite was analyzed by determination of cell yield, osteocalcin production, and levels of alkaline phosphatase. The viable cells that were retrieved from the GPEEK discs of all three surface roughness had an approximate sixfold increase in number. Osteoblastic function of the cells, indicated by osteocalcin production, was unimpaired after a 5-day culture on the three surfaces of GPEEK. The highest level of osteocalcin was produced by osteoblastic cells propagated on GPEEK with a 9 microns surface roughness. The levels of alkaline phosphatase of these cells were similarly greater for the different degrees of surface roughness. Overall, this study demonstrates that GPEEK supported proliferation of osteoblastlike cells and provided a favorable environment for the continued production of osteocalcin in vitro.

Inhibition of cyclooxygenase-2 expression and prostaglandin E2 production in chondrocytes by avocado soybean unsaponifiables and epigallocatechin gallate

OBJECTIVE To evaluate the anti-inflammatory effect of the combination of avocado soybean unsaponifiables (ASU) and epigallocatechin gallate (EGCG) on cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) production in cytokine-activated equine chondrocytes. METHODS Production of type II collagen and aggrecan was verified by immunohistochemistry and Western blot. Chondrocytes were incubated with: (1) control media alone, (2) ASU (4 microg/ml; 8.3 microg/ml), (3) EGCG (4, 40, 400 ng/ml), or (4) the combination of ASU and EGCG for 24h. Cells were next incubated with control medium alone or with IL-1beta (10 ng/ml) and TNF-alpha (1 ng/ml). COX-2 gene expression by real-time PCR analysis and NF-kappaB nuclear translocation by immunohistochemistry were performed after 1h of incubation. PGE(2) production was determined by immunoassay after 24h of incubation. RESULTS Equine chondrocytes responded to cytokine activation by up-regulated gene expression of COX-2 and increased PGE(2) production. Activation was associated with NF-kappaB translocation. Individually, ASU and EGCG marginally inhibited COX-2 expression and PGE(2) production in activated chondrocytes. In contrast, the combination of ASU and EGCG reduced COX-2 expression close to non-activated control levels and significantly inhibited PGE(2) production. These reductions were statistically greater than those of ASU or EGCG alone. The inhibition of COX-2 expression and PGE(2) production was associated with inhibition of NF-kappaB translocation. CONCLUSION 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.

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.

Differential effects of reserpine on brainstem catecholaminergic neurons revealed by Fos protein immunohistochemistry

The effects of a single systemic injection of reserpine on c-fos proto-oncogene expression in catecholaminergic neurons of the rat brainstem were studied by immunohistochemistry for Fos proteins (Fos). In control rats, a few Fos immunoreactive neuronal nuclei were observed in the tectum and mesencephalic central gray. Within hours after drug injection, a substantial number of brainstem neurons stained intensely for Fos. The staining was maximal at 6 h and returned to control levels within 24 h. Double-immunohistochemical staining with antibodies to tyrosine hydroxylase revealed that in all noradrenergic (NA) neuron subgroups except the A2 group, the majority of NA neurons stained for Fos. Most adrenergic neurons were also labeled. In contrast, aside from some cells in the ventral tegmental area, reserpine did not induce Fos immunoreactivity in dopaminergic neurons. Numerous non-catecholaminergic neurons were intensely stained with Fos in the substantia nigra pars reticulata, ventral tegmental area, mesencephalic central gray, pontine nuclei and tectum. A small number of Fos immunoreactive neurons was also observed in raphe nuclei. Injection of saline (i.p.) resulted in a moderate increase in Fos immunoreactivity in the locus ceruleus, in A1/C1 neurons and in the mesencephalic central gray. The results demonstrate that acute reserpine treatment induces Fos expression in distinct populations of brainstem neurons, comprising both catecholaminergic and non-catecholaminergic neurons. Thus, induction of Fos by reserpine does not coincide with the site of action of this drug. The distribution of Fos immunoreactive NA neurons after reserpine treatment is comparable to that reported after application of stressful stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Proinflammatory cytokine expression of IL‐1β and TNF‐α by human osteoblast‐like MG‐63 cells upon exposure to silicon nitride in vitro

This study was designed to determine the effect of Si(3)N(4) disks and particulates on human osteoblast-like MG-63 cells in vitro. The MG-63 (10(5)/mL) cells were plated onto 24-well polystyrene plates fitted with either sintered reaction-bonded (SRBSN) or reaction-bonded (RBSN) 15-mm disks. Controls consisted of wells without Si(3)N(4) disks. Cells propagated at 37 degrees C, 5% CO(2) for 48 h on Si(3)N(4) disks and control polystyrene surfaces exhibited similar proliferative capacities (7000 and 4000 cpm/10(5) cells, respectively, p > 0.05). Cells incubated with 1, 10, or 100 microgram/ml of Si(3)N(4) particles (<1.00 to 5.00 micrometer) for 24 h did not exhibit a decrease in DNA synthetic activity: 12 +/- 1.3 x 10(4), 10.5 +/- 1.5 x 10(4), and 11.0 +/- 1.7 x 10(4) cpm, respectively, compared to 11.6 +/- 2.6 x 10(4) cpm/10(5) for the control cells, as indicated by (3)H-thymidine uptake. Cells propagated on RBSN displayed increased expression of cytokines IL-1beta and TNF-alpha compared to the control cells, as shown by reverse transcriptase-polymerase chain reaction (RT-PCR). In contrast, cells propagated on SRBSN surfaces expressed the same level of IL-1beta and TNF-alpha as that of control cells. Incubation of MG-63 cells with 1-10 microgram/mL of particles did not increase IL-1beta expression. However, at 100 microgram/mL, TNF-alpha expression was greater than that of the control cells. Silicon nitride, evaluated here as disks or as particulates (1-10 microgram/mL), is biocompatible and does not hinder the proliferation or induce proinflammatory cytokine expression of human osteoblast-like MG-63 cells in vitro.