Proliferation Of Articular Chondrocytes Research Articles

CDNA cloning and chromosomal mapping of rat Smad2 and Smad4 and their expression in cultured rat articular chondrocytes.

Smad proteins are known to transduce signalling of TGF-beta receptor superfamily. We report here the entire sequences of rat Smad2 and Smad4 which have not been identified yet. Entire sequences were identified by degenerated polymerase chain reaction and following phage library screening and 5' RACE. The predicted amino acid sequences of rat Smad2 and Smad4 are highly conserved among rat, human and mouse. We also mapped these Smads to chromosome 18q.12.3. Unlike endothelial cells, TGF-beta1 stimulates articular chondrocyte proliferation as well as extracellular matrix production, and acts as a repairing agent against cartilage destruction. Since both Smad2 and Smad4 are essential factors for TGF-beta signalling, we examined their expression and regulation in cultured articular chondrocytes. Northern blot analysis showed that TGF-beta1 significantly increased the mRNA level of Smad2 but not of Smad4 in a dose- and time-dependent manner, suggesting that the augmentation of TGF-beta1 action is caused by increasing the expression of the downstream signalling molecule.

Articular chondrocytes and synoviocytes in culture: influence of antioxidants on lipid peroxidation and proliferation

Chondrocytes and synoviocytes are the main cell types in articular joints. Articular cartilage is fed by synoviocytes via synovial fluid and has a low partial oxygen pressure. Thus, chondrocytes show oxygen radical protective mechanisms in vivo and are unprotected against these factors under common culture conditions. We investigated the influence of ascorbic acid, Fe2+, glutathione and alpha-tocopherol on lipid peroxidation and proliferation of rat articular chondrocytes and rabbit synoviocytes (HIG-82) in vitro. A combination of ascorbic acid and Fe2+ induced the production of thiobarbituric acid-reactive material as a marker of radical-mediated lipid peroxidation in homogenates and/or supernatants of cultured chondrocytes and synoviocytes. The amount of lipid peroxidation of chondrocytes was about 3-fold higher than that of synoviocytes. Ascorbic acid or Fe2+ alone had no significant influence on the production of thiobarbituric acid-reactive material. Lipid peroxidation could be abolished by addition of the radical scavenger alpha-tocopherol, whereas glutathione had no effect. 25-50 microM alpha-tocopherol decreased the ascorbic acid-(100 micrograms/ml) and Fe(2+)-(3 microM) induced lipid peroxidation to a basal level. Moreover, ascorbic acid inhibited the proliferation of rat chondrocytes and rabbit synoviocytes measured by [3H]-thymidine incorporation. Alpha-tocopherol and glutathione had no influence on the proliferation of chondrocytes but alpha-tocopherol decreased the growth of synoviocytes and increased the anti-proliferative effect of ascorbic acid on these cells. The importance of these findings for the use of ascorbic acid, glutathione and alpha-tocopherol in chondrocyte and synoviocyte cultures, or the influence of these molecules on the etiology and treatment of articular diseases will be discussed.

Effects of IGF-I, IGF-II, and PDGF on the proliferation of rabbit articular chondrocytes in culture

The effects of insulin-like growth factor (IGF)-I, IGF-II, and platelet-derived growth factor (PDGF) were examined in cultured chondrocytes derived from rabbit joint cartilage. In addition, the expression of IGF-I receptors on the cultured chondrocytes was detected with anti-IGF-I receptor antibody. IGF-I acted as a growth stimulant, but IGF-II had no effect on cell growth. Increasing the dose of IGF-I beyond 50ng/ml resulted in decreased growth stimulation. The expression of IGF-I receptors was detected continuously during the culture period. The results also showed that PDGF acted as a growth inhibitor, in contrast to the results of other studies. Further studies are necessary to clarify the precise mechanism of the action of PDGF and the role played by different homoor heterodimers of PDGF in the proliferation of articular chondrocytes.

TGF-βPromotes the Growth of Bovine Chondrocytes in Monolayer Culture and the Formation of Cartilage Tissue on Three-Dimensional Scaffolds

We have investigated the ability of transforming growth factor beta (TGF-beta) to promote the growth and differentiation of chondrocytes in monolayer and on three-dimensional scaffolds. Treatment of chondrocytes with TGF-beta and ascorbate individually stimulated the proliferation of bovine articular chondrocytes about 2-fold when cells were grown in monolayer culture: the combination of TGF-beta and ascorbate resulted in a 3-fold increase in cell number over a 72-h period. Peak stimulation with TGF-beta occurred at about 1.0 ng/ml: bFGF was slightly inhibitory in these assays. TGF-beta led to an increase in glycosaminoglycan synthesis as detected by Western blotting using anti-chondroitin sulfate antibodies. No significant change in collagen type II mRNA or protein was observed. When cells were grown on grown on three-dimensional scaffolds composed of polyglyocolic acid, TGF-beta treatment led to an increase in the size of the cartilage-like constructs produced. This was accompanied by increases in collagen and glycosaminoglycan deposition; immunohistochemical staining showed that the predominant collagen was type II. These results indicate that TGF-beta is capable of increasing the proliferation rate of chondrocytes in monolayer as well as increasing cartilage production on three-dimensional scaffolds and may find utility in the in vitro engineering of cartilage tissue.

Differentiation-dependent effects of IL-1 and TGF-beta on human articular chondrocyte proliferation are related to inducible nitric oxide synthase expression.

This study analyzed the effect of chondrocyte differentiation on iNOS expression and responses to IL-1 and TGF-beta. During subculturing of chondrocytes, the growth-stimulatory effects of TGF-beta decreased, and cells in later passages even were growth inhibited by TGF-beta. IL-1 beta responses showed an inverse pattern. The antiproliferative effects of IL-1 beta decreased, and, after passage 6, IL-1 beta became a growth stimulator for chondrocytes. This change in growth factor response pattern was associated with a decrease in type II collagen expression. To determine whether these changes in the growth regulatory effects of IL-1 beta and TGF-beta were related to nitric oxide (NO), inducible nitric oxide synthase (iNOS) expression and NO release were analyzed. In primary chondrocytes, TGF-beta did not stimulate iNOS mRNA expression or NO release, and, during co-incubation, it did not detectably alter the IL-1 beta effect. Preincubation with TGF-beta resulted in a time-dependent increase in IL-1-induced NO. With increasing passage number, the IL-1 beta effects decreased, and, after passage 6, IL-1 beta no longer detectably stimulated iNOS expression or NO release. However, TGF-beta increased NO production synergistically with IL-1 beta during the same culture period when it lost its growth-stimulatory effects. The antiproliferative effects of TGF-beta in late passage chondrocytes were reversed by the NO synthase inhibitor NG-monomethylarginine. These results suggest a novel pattern of iNOS regulation by IL-1 and TGF-beta and show that the factors that modulate iNOS expression and proliferation are dependent on the differentiation status of the cells.

Modulation of rabbit articular chondrocyte (RAC) proliferation by TGF-beta isoforms.

We have previously shown that TGF-beta 1 exerts a bifunctional effect on RAC proliferation. Added to quiescent cultures, it inhibits the entry of G0/G1 cells into S phase whereas in S phase synchronized populations, it stimulates the DNA replication rate with a delayed G2 + M phase and a subsequent transient increase of cell number. As TGF-beta 2 and beta 3 isoforms are also expressed in bone and cartilage tissues, it was of interest to study their effect on RAC proliferation, in comparison to that of TGF-beta 1. Using cell counting and tritiated thymidine incorporation, we found that all the TGF-beta s used here induced an increase of RAC proliferation rate occurring between 24 and 48 h of exposure. TGF-beta 2 appeared as the most efficient form as judged from the maximum of thymidine labelling. However, TGF-beta 3 induced an increase of cell number slightly higher than both TGF-beta 1 and TGF-beta 2 (+30% versus 20% for TGF-beta 1 and beta 2). TGF-beta 2 and beta 3 were able to stimulate the DNA replication rate as previously demonstrated for TGF-beta 1. However, the effect occurred later for TGF-beta 2 and beta 3 (12 h) than for TGF-beta 1 (6 h). This was confirmed by flow cytometric analysis of DNA content. In addition, immunodetection by flow cytometry demonstrated that all TGF-beta isoforms enhanced endogenous expression of TGF-beta-related peptides. The effect was shown to be associated with the cell cycle S phase and was greater for TGF-beta 3 than for TGF-beta 1 and beta 2. These findings suggest that TGF-beta s could act on RAC functions via autocrine and paracrine ways. Taken together, these data indicate that TGF-beta s may modulate proliferation of articular chondrocytes and therefore could play a role in the activation of these cells in the early stages of osteoarthritis.

Growth hormone directly and indirectly stimulates articular chondrocyte cell growth

Although growth hormone (GH) is known to regulate cartilage growth and differentiation during development, it is still unclear whether the cell growth of articular chondrocytes is stimulated directly by GH or mediated by GH-induced insulin-like growth factor-I (IGF-I). In the present study, we focused on whether GH directly or indirectly stimulates articular chondrocyte proliferation. Monolayer articular chondrocytes from 5-week-old male Sprague-Dawley rats were cultured in Ham's F-12/Dulbecco's modified essential medium supplemented with 10% fetal bovine serum. Stimulation of DNA synthesis by GH was dose-dependent between 0.1 and 1 microg/ml, and the maximum active concentration of GH was 500 ng/ml, which induced a 3.5-fold increase over control values. Anti-IGF-I antiserum neutralized about 80% of GH-induced DNA synthesis. GH stimulated the secretion of IGF-I into the conditioned medium in a dose-responsive manner. To determine whether GH stimulated DNA synthesis directly, we investigated the time-course changes in mRNA expression of IGF-I and the proto-oncogene c-myc. Induction of IGF-I mRNA occurred at 4 h, and reached a maximum level at 12 h, whereas the expression of c-myc mRNA was induced within 4 h, and continued to increase until 72 h after GH treatment. Furthermore, administration of cycloheximide, an inhibitor of protein synthesis, resulted in the superinduction of both IGF-I and c-myc mRNAs. These results suggest that early induction of c-myc is due to a direct stimulatory effect of GH, and that long-term induction of c-myc was attributable to an indirect effect of GH in which GH-induced secondary proliferative factors may act in an autocrine/paracrine manner. The superinduction of c-myc gene by cycloheximide also indicates that fresh protein synthesis of an intermediate protein was not required for GH-induced c-myc expression. Western ligand blot analysis of IGF-binding proteins revealed that cultured rat articular chondrocytes produced a predominant 41 kDa and a faint 32 kDa form, and that GH significantly stimulated the secretion of the 41 kDa form without affecting expression of the 32 kDa form. Furthermore, a specific IGF-I binding study suggested that the increase in DNA synthesis induced by GH was not associated with changes in affinity or in the number of IGF-I binding sites. These results support the conclusion that the stimulatory effect of GH was mainly mediated by GH-induced IGF-I production in monolayer rat articular chondrocytes. However, it is likely that GH may also have a direct stimulatory effect by inducing c-myc proto-oncogene expression.

Effects of transforming growth factor β and interleukin-1β on [ 3H]thymidine incorporation by human articular chondrocytes in vitro

This is a study of the regulation of human articular chondrocyte proliferation by transforming growth factor β (TGFβ) and interleukin-1β (IL-1β) in vitro. Human articular chondrocytes were cultured at different cell densities on plastic and on a collagen substratum, in the presence and absence of serum. The effects TGFβ amd IL-1β on proliferation of chondrocytes, as determined by [ 3H]thymidine incorporation, under these conditions of culture were examined. TGFβ was found to have both stimulatory and inhibitory effects on chondrocytes in vitro. Interactions between TGFβ and growth factors present in serum influence the modulation of chondrocyte proliferation by TGFβ. IL-1β caused a significant reduction of the TGFβ-stimulated increase in chondrocyte proliferation. The complex inter-relationships between TGFβ and IL-1β on chondrocytes have implications for cartilage repair.

IPG (inositolphosphate glycan) as a cellular signal for TGF‐β1 modulation of chondrocyte cell cycle

The knowledge of transforming growth factor (TGF)-beta receptors has greatly progressed in the recent years. TGF-beta receptors type I and II have been implicated in the modulation of cell proliferation, whereas type III (betaglycan) may act as a component presenting TGF-beta to its signaling receptors. In addition, four other proteins that bind TGF-beta 1 or TGF-beta 2 have been recently identified in some cell lines, three being anchored to the membrane through a glycosylphosphatidylinositol (GPI). Despite this knowledge, the molecular mechanism of signal transduction through the TGF-beta receptors remain an enigma. TGF-beta family does not signal via any of the classical pathways. As GPI anchors of membrane proteins have been implicated in the transduction of some hormonal effects, we investigated the putative role of GPI in signaling the TGF-beta effects on the proliferation of rabbit articular chondrocytes (RAC). We previously showed that TGF-beta 1 increased DNA replication rate of RAC, with a recruitment of cells in G2/M followed by a subsequent mitosis wave. Here, we find that the factor causes specific GPI hydrolysis, with correlated increase of inositolphosphate glycan (IPG). This effect was specifically inhibited by antibodies that bind TGF-beta 1. Using [3H]-inositol labeling and Triton X-114 extraction, we demonstrate that a hydrophobic material from the membrane is cleaved by treatment of cell cultures with phosphatidylinositol specific phospholipase C (PI-PLC) or by exposure to TGF-beta, supporting that a PI-anchored molecule gives rise to IPG by TGF-beta-induced hydrolysis. The biological relevance of this hydrolysis was demonstrated by the enhancing effect of purified IPG on the DNA synthesis rate, which mimicked the TGF-beta action. These results demonstrate that IPG could be an early messenger in the cellular signaling that mediates the effect of TGF-beta on RAC growth.

The cells from rat aorta scrapings (RAS) synthesize factor(s) that is (are) very potent stimulator(s) of rat articular chondrocytes proliferation (RACP) and proteoglycan and collagen synthesis

The cells from rat aorta scrapings (RAS) synthesize factor(s) that is (are) very potent stimulator(s) of rat articular chondrocytes proliferation (RACP) and proteoglycan and collagen synthesis

PMA enhances IGF-1 and EGF but has no effect on PDGF- and FGF β-induced stimulation of rat articular chondrocyte proliferation

PMA enhances IGF-1 and EGF but has no effect on PDGF- and FGF β-induced stimulation of rat articular chondrocyte proliferation

Comparative effects of growth factors and cytokines on rat articular chondrocyte proliferation

Comparative effects of growth factors and cytokines on rat articular chondrocyte proliferation

Flow cytometric detection of transforming growth factor-β expression in rabbit articular chondrocytes (RAC) in culture—Association with S-phase traverse

We have previously shown that TGF-β1 decreased the entry of G0 G1 -synchronized rabbit articular chondrocytes (RAC) into S-phase, whereas it enhanced the proliferation rate of actively dividing cells (asynchronous or S-phase-synchronized cells). The growth proliferative effect was accompanied by both increased DNA replication rate and G2 M delay. Since TGF-β mRNA has been detected in chondrocytes, it was of interest to study the expression of the factor in correlation with the cell cycle of RAC. Using cytofluorometric analysis of both DNA content and TGF-β protein level, we demonstrated that S-phase-synchronized RAC constitutively expressed TGF-β, whereas G0 G1 -synchronized cells only display very low levels of the factor. The data showed that the expression of TGF-β is correlated with S-phase traverse since it increases with the percentage of cells in S-phase (less than 27% in G0 G1 to 70% in S-phase-synchronized cells). Moreover, exposure of RAC to TGF-β1 (1 ng/ml) for 24 h increased the percentage of positive cells, independently of the number of cells in S-phase, indicating that the factor may up-regulate its own expression. All together, these data suggest that TGF-β could play a role in initiating the proliferation of articular chondrocytes during the early events of osteoarthritis and might take a part in the repair of cartilage matrix.

IL-1β-induced expression of PDGF-AA isoform in rabbit articular chondrocytes is modulated by TGF-β1

Interleukin 1β (IL-1β) and platelet-derived growth factor (PDGF) induce proliferation in many cell types. Both peptides are released by activated macrophages and other cells in response to injury and are thought to play a crucial role in a number of pathological processes. We found that IL-1β stimulates proliferation of rabbit articular chondrocytes and induces synthesis and release of PDGF into their culture medium. This effect, which is time- and dose-dependent (0.05–5 ng/ ml), is restricted to PDGF-AA, one of the three PDGF isoforms; IL-1β effect on PDGF is inhibited by actinomycin D and α-amanitin, suggesting a transcriptional regulation of PDGF-A chain. IL-1β stimulates PDGF-AA synthesis also in the presence of indomethacin, a prostaglandin synthesis inhibitor. Transforming growth factor β1 (TGF-β1), a dimeric polypeptide which displays multiple biological activities, inhibits in a dose-dependent manner (1–10 ng/ml) PDGF-AA production induced by IL-1β. In a binding assay, TGF-β1 induces 45% decrease in specific binding sites for 125I-IL-1β, with no change in affinity.

Time-varying magnetic fields: effects of orientation on chondrocyte proliferation.

The purpose of this study was to determine the effect of orientation of pulsed electromagnetic fields (PEMFs) on cellular proliferation and extracellular matrix synthesis. Bovine articular chondrocytes were cultured in PEMFs (repetitive pulse at 72 Hz) generated using Helmholtz coils oriented either parallel (horizontal) or perpendicular (vertical) to the plane of cell adhesion. Dissipation of signal energy in the form of heat increased the temperature of the PEMF coils by 2 degrees C and the tissue culture medium by 1 degree C. Therefore, control coils, which emitted no PEMFs, were heated to the temperature of PEMF coils by circulating water. Chondrocytes were cultured in 16-mm-well culture plates, and the data for individual wells were pooled as triplicates. Although not observed by microscopic examination of individual wells, positionally dependent electric field effects may be minimized by this approach. PEMFs generated by coils oriented vertically significantly decreased chondrocyte proliferation. The effect was dependent on the concentration of serum in the culture media. At 3% serum concentration, the total cell number attained after 10 days of culture was reduced by 50% in stimulated cultures when compared with controls. At 5% serum concentration, there was no effect. PEMFs applied by coils oriented horizontally did not alter proliferation of articular chondrocytes. PEMFs had no effect on synthesis of extracellular matrix by chondrocytes plated at high density, irrespective of orientation.

Rabbit articular chondrocytes in soft agar are useful to test the effect of non-steroidal anti-inflammatory drugs on chondrocyte replication.

The effect of non-steroidal anti-inflammatory drugs (NSAIDs) on rabbit articular chondrocyte proliferation was examined in soft agar and in conventional cultures on plastic dishes. Indomethacin, acetylsalicylate, and naproxen (10(-4) M) decreased the efficiency of colony formation by chondrocytes in soft agar in the presence of fibroblast growth factor, although the drugs had little effect on the proliferation of chondrocytes on plastic dishes. On the other hand, tiaprofenic acid (10(-4) M) did not have any adverse effect on chondrocyte replication in either culture the two systems. Rabbit articular chondrocyte growth in soft agar will be a useful test system for examining the effects of anti-arthritic drugs on cartilage metabolism and repair.

Effects of dexamethasone on the growth of cultured rabbit articular chondrocytes:relation with the nuclear glucocorticoid-receptor complex.

This study reports that dexamethasone at a high dose (10(-4) mol/l) induced slowing of the in vitro proliferation of rabbit articular chondrocytes in both monolayer and clonal culture. This effect is consistent with an inhibition of DNA and RNA synthesis and was characterised by an accumulation of cells in the G0G1 phase of the cell cycle, as shown by flow cytometric analysis. Therefore we determined the extent of nuclear localisation of dexamethasone-receptor complexes. The results showed a discrepancy between 50% growth inhibitory dose (10(-4) mol/l) and the apparent affinity, KD (1.4 (SD 0.2) X 10(-9) mol/l). Thus the growth inhibition of rabbit articular chondrocytes by dexamethasone did not seem to be related exclusively to an interaction with the glucocorticoid-receptor complexes.

97 HUMAN FETAL AND ADULT CHONDROCYTES: CLONAL GROWTH PROMOTION BY INSULIN LIKE GROWTH FACTORS I AND II, INSULIN AND GROWTH HORMONE

A direct assay for cellular growth was used. It measures the capacity of isolated chondrocytes to proliferate in a clonal manner starting from a single cell suspension in a semisolid system (0.8% methylcellulose, BM Whissler Medium, 5% heat inactivated serum, 1000 inserted chondrocytes/ml). This assay was used to measure the clonal proliferation of human fetal (20th week of pregnancy, n=10) and adult articular chondrocytes 20yrs, n= 10) in response to insulin like growth factors I and II (IGF I/II), biosynthetic human insulin (BHI) and human growth hormone (hGh). Both BHI (100 ng/ml) and hGh (100 ng/ml) did not stimulate fetal and adult chondrocytes to proliferate in a clonal manner. IGF I (25 ng/ml) stimulated clonal growth of fetal chordrocytes (60 ± 12 colonies/1000 inserted cells; m ± 1 SD), however IGF II (25 ng/ml) was significantly more effective (116 ± 12 colonies/1000 inserted cells; p < 0.05). In contrast IGF I was more effective in adult chondrocytes (40 ± 5 colonies/1000 inserted cells) than IGF II (24 ± 2 colonies/1000 inserted cells; p < 0.05).

The effect of soluble sodium urate on the proliferation and proteoglycan synthesis of lapine articular chondrocytes in monolayer culture.

The effects of soluble monosodium urate (350, 50, and 5 mumol/l) on the proliferation and proteoglycan synthesis of lapine articular chondrocytes were studied in a monolayer culture system. Cellular proliferation in vitro was unaffected by urate treatment. Chondrocytes treated with 50 mumol/l sodium urate showed a 26% reduction in binding of 35SO4 to matrix macromolecules. However, this reduction did not achieve statistical significance. The 350 and 5 mumol/l concentrations did not alter 35SO4 binding. It is proposed that monosodium urate has no marked direct effect on chondrocyte viability, proliferation and proteoglycan synthesis.