The chondrocyte is the unique cell type in articular cartilage. It is responsible for the extracellular matrix production which permits the cartilaginous tissue to compensate the skeletal pressures during movements. The regulation of the different extracellular matrix components (type II collagen and aggrecan) depends on the differentiated state of the chondrocyte. However, in vivo, such as during aging and osteoarthritis, as well as in vitro, in monolayer culture, chondrocytes lose their morphological and biochemical characteristics. This phenomenon has been particularly studied in culture. As soon as at passage 1, there is a gradual shift from the synthesis of type II collagen to type I and III collagens and from the synthesis of large aggregating proteoglycans (aggrecan) to low molecular weight proteoglycans. However, it has been shown that dedifferentiated chondrocytes can reexpress phenotypic markers of the articular chondrocyte. These conditions include tridimensional culture in gels of agarose, collagen and alginate. This can be also obtained after treatment of chondrocytes in monolayer by dihydrocytochalasin B or staurosporine. Although the mechanisms involved in restoration of the differentiated phenotype have not been elucidated yet, it has been shown that the synthesis of specific proteoglycans and type II collagen can be related to a modification of actin architecture. The restoration of the differentiated functions using tridimensional culture of chondrocytes has been used to perform autologous chondrocyte implantation in chondral defects. In the future, the grafts could be improved using chondrocytes treated with stimulating growth factors and synthetically derived matrices.

The chondrocyte is the unique cell type in articular cartilage. It is responsible for the extracellular matrix production which permits the cartilaginous tissue to compensate the skeletal pressures during movements. The regulation of the different extracellular matrix components (type II collagen and aggrecan) depends on the differentiated state of the chondrocyte. However, in vivo, such as during aging and osteoarthritis, as well as in vitro, in monolayer culture, chondrocytes lose their morphological and biochemical characteristics. This phenomenon has been particularly studies in culture. As soon as at passage 1, there is a gradual shift from the synthesis of type II collagen to type I and II collagens and from the synthesis of large aggregating proteoglycans (aggrecan) to low molecular weight proteoglycans. However, it has been shown that dedifferentiated chondrocytes can reexpress phenotypic markers of the articular chondrocyte. These conditions include tridimensional culture in gels of agarose, collagen and alginate. This can be also obtained after treatment of chondrocytes in monolayer by dihydrocytochalasin B or staurosporine. Although the mechanisms involved in restoration of the differentiated phenotype have not been elucidated yet, it has been shown that the synthesis of specific proteoglycans and type II collagen can be related to a modification of actin architecture. The restoration of the differentiated functions using tridimensional culture of chondrocytes has been used to perform autologous chondrocyte implantation in chondral defects. In the future, the grafts could be improved using chondrocytes treated with stimulating growth factors and synthetically derived matrices.

Immortalization of chondrocytes by SV40 T Ag has often been reported to trigger the loss of expression of type II collagen, one of the main differentiation markers, although some immortalized chondrocyte lines maintaining a differentiated phenotype have also been described. Here, we show using transient cotransfections in differentiated chondrocytes that, in contrast to c-src, neither SV40 T Ag, nor c-myc, decreases col2a1 transcriptional activity. Then, we report the possibility of immortalizing rabbit articular chondrocytes by expression of SV40 T Ag controlled by the col2a1 promoter and enhancer (pCol2SV). This strategy allows one to select within a population of differentiated chondrocytes those which are able to maintain functional regulation of the col2a1 gene through long-term culture. In precrisis pCol2SV-transfected chondrocytes, all-trans-retinoic acid, a down-regulator of col2a1 expression, induced apoptosis, strongly suggesting the strict control of T Ag expression by col2a1 regulatory sequences. Some pCol2SV-transfected chondrocytes were definitively immortalized, after a short crisis period. However, type II collagen synthesis was restricted to a small proportion of cells, which went on to decrease with subculture, while the proportion of cells expressing T Ag was not affected. In these postcrisis cells, T Ag remained at least partially under the control of functional col2a1 regulatory elements as assessed by all-trans-retinoic acid down-regulation.

Tendinitis and tendon rupture complicating fluoroquinolone therapy have been reported recently, especially affecting men over 60 years. These new quinolones are more potent antimicrobial agents than older nonfluorinated compounds like nalidixic acid. We compared the effects of one quinolone (nalidixic acid) and two fluoroquinolones (norfloxacin and pefloxacin) on cultured rabbit Achilles tendon cells. First, we examined their effects on cell viability, mitochondrial succinate dehydrogenase and global activity, mitochondrial activity using microtitration methods. Pefloxacin and norfloxacin were more cytotoxic than nalidixic acid according to IC50 values. These results confirm that mitochondria represent a biological target of fluoroquinolones. Moreover, the extracellular matrix was studied by molecular hybridization. After a 72 h treatment, the level of type I collagen transcripts was not modified with any of the three antimicrobial agents, whereas mRNA encoding decorin was decreased with 10-4 mol/L pefloxacin only. The decrease of transcripts encoding decorin suggests that this matrix component is another target of pefloxacin and modification of decorin seems to be an early event (before mitochondrion alteration) which may contribute to the explanation of tendon rupture.

Understanding the mechanisms responsible for photodamage to the skin is most important for dermatology, 3-D cultures have been used as tools to mimic the in vivo situation for several years. We irradiated such a system containing human dermal fibroblasts cultured in collagen gels, a well-known model considered to be a dermal equivalent, which reproduces the interaction between cells and the surrounding extracellular matrix. The effects of solar irradiation (315–800 nm) on the stead-state levels of the mRNAs of extracellular matrix components (type 1 and III collagens) and their degrading enzymes (interstitial collagenase, MMP-1 and stromelysin 1, MMP-3) were measured. Exposure to low levels of solar radiation (0–10 J cm 2 in the UVA, i.e. suberythemal UVA doses) caused a transient decrease in type 1 procollagen mRNA, an increase in MMP-mRNA, and no change in type III procollagen mRNA steady-state levels. These results describe the early changes in the connective tissue of the skin following exposure to low level solar stimulation, and may help explain the long-term changes in photodamaged skin.

Chondrocytes cultivated in monolayer rapidly divide and lose their morphological and biochemical characteristics, whereas they maintain their phenotype for long periods of time when they are cultivated in alginate beads. Because cartilage has a low cellularity and is difficult to obtain in large quantities, the number of available cells often becomes a limiting factor in studies of chondrocyte biology. Therefore, we explored the possibility of restoring the differentiated properties of chondrocytes by cultivating them in alginate beads after two multiplication passages in monolayer. This resulted in the reexpression of the two main markers of differentiated chondrocytes: Aggrecan and type II collagen gene expression was strongly reinduced from day 4 after alginate inclusion and paralleled protein expression. However, 2 weeks were necessary for total suppression of type I and III collagen synthesis, indicators of a modulated phenotype. Interleukin-1beta, a cytokine that is present in the synovial fluid of rheumatoid arthritis patients, induces many metabolic changes on the chondrocyte biology. Compared with cells in primary culture, the production of nitric oxide and 92-kDa gelatinase in response to interleukin-1beta was impaired in cells at passage 2 in monolayer but was fully recovered after their culture in alginate beads for 2 weeks. This suggests that the effects of interleukin-1beta on cartilage depend on the differentiation state of chondrocytes. This makes the culture in alginate beads a relevant model for the study of chondrocyte biology in the presence of interleukin-1beta and other mediators of cartilage destruction in rheumatoid arthritis and osteoarthrosis.

Following multicentre studies and ECVAM proposals, microtitration tests have been recommended for use in screening protocols in cellular pharmacotoxicology. The advantages of these tests are their good standardization and reproducibility, but they are often a straightforward extrapolation of biochemical methods, which require the initial extraction of the marker studied. Consequently, detection specificity is low and sensitivity is only moderate, so that adaptation to cellular heterogeneity is difficult. By contrast, the use of laser excitation in flow cytometry methods (FCM), with fluorescent probes, ensures the specific and sensitive detection of intracellular markers. However, these costly and difficult methods are not suitable for general screening purposes.The aim was to combine the benefits of both types of methods in order to obtain microplate cytometric methods with three characteristics: specificity, sensitivity (pg/ml) and standardization. A new cold light fluorimetry technology (CLF) was therefore developed and adapted for use with microplates. This microplate cytofluorimetry (MCM) can be used directly for microtritration fluorimetric assays on alive cells (MiFAAC).These MiFAAC tests are characterized by Various points: specificity of the reaction due to the use of low-cost fluorescent or fluorigenic probes; sensitivity of detection due to the use of cold light micro titration fluorimetry (pg/ml); standardization of the protocols with microplates or Petri dishes; very wide detection spectrum: 280-870 nm. As these tests are performed on living cells, which permit kinetic studies, a single support (a microplate) can be used for the three steps of the test: 1) cell culture, 2) operating protocol, and 3) detection with the fluorescent probe read directly by using a microplate cytofluorimeter (Fluorolite 1000, Dynex(TM)), which incorporates the cold light fluorimetry.Moreover, MiFAAC tests permit different biological endpoints to be studied for cytotoxicity evaluation as recommended by ECVAM, e.g. Hoechst 33342 probe/DNA-cell proliferation, neutral red uptake using fluorimetric detection/cell viability, and rhodamine 123/mitochondrial activity These UV, visible, red probes are used with high sensitivity and detection limits (2, 20 and 50 pg/ml, respectively).The autofluorescence of different types of microplates has been evaluated. Clear microplates revealed a high autofluorescence, especially with UV excitation. White microplates were not suitable for fluorimetry. Black microplates have low autofluorescence, but cell microscopy cannot be performed. So, new microplates with black sides and a clear flat bottom are completely adapted to microplate cytofluorimetry, due to their low autofluorescence with UV, green visible and red excitation. Moreover, cell monitoring can be performed with microscopy or image analysis. These MiFAAC tests therefore allow the direct use of numerous fluorescent probes on living cells, so that sets of tests can be assembled.These rapid tests can be associated in preliminary phases with flow cytometry or image analysis on microplates. Their specificity, sensitivity (pg/ml) and standardisation ensure that they are well-adapted to cellular heterogeneity and comply with the requirements of cellular pharmacotoxicological screening procedures.

The culture of rabbit tenocytes could be a useful model in the study of the physiopathology and pharmacotoxicology of tendons. This work was undertaken to examine the in vitro behavior of tenocytes from juvenile rabbit Achilles tendons. We report observations of the morphological and biological characteristics of primary culture and subsequent passages of rabbit tendon cells cultured in monolayer. Data obtained by electron microscopy and growth curves were complementary. After 36 passages, the generation time of tenocytes did not change and no sign of senescene could be seen. Primary culture and the first passages retained the expression of tenocyte differentiated functions, synthesis of type I collagen and decorin. Cell growth behavior was not modified upon passaging. However, when subcultured, tenocytes displayed a modulated phenotype.

Several studies were undertaken to develop three-dimensional (3-D) cell culture models that allow conditions closer to the in vivo situation. To this end, alginate gels were tested as a 3-D cell culture model that might be useful in the study of the effects of UVA on human dermal fibroblasts. Cell culture in alginate gels and the irradiation conditions were optimized. Results showed that optimized cultures in alginate gels experienced considerable cell death on UVA irradiation compared to the classical monolayer cell culture. Viability tests (cell counting and neutral red assay) were performed to show that only UVA-irradiated alginate gels were responsible for this cytotoxicity. The implication of oxygen species in the phototoxicity induced by ultraviolet light has already been described; for this reason we investigated whether oxygen species were involved in the cytotoxicity induced by alginate upon UVA irradiation. It appeared that superoxide anion is not implicated.