Phenotypic diversity of neoplastic chondrocytes and extracellular matrix gene expression in cartilaginous neoplasms.

Chondrocyte differentiation is characterized by distinct cellular phenotypes, which can be identified by specific extracellular matrix gene expression profiles. By applying in situ analysis on the mRNA and protein level in a series of benign and malignant human chondrogenic neoplasms, we were able to identify for the first time different phenotypes of neoplastic chondrocytes in vivo: 1) mature chondrocytes, which synthesized the characteristic cartilaginous extracellular tumor matrix, 2) cells resembling hypertrophic chondrocytes of the fetal growth plate, 3) cells resembling so-called dedifferentiated chondrocytes, and 4) well differentiated chondrocytic cells, which expressed type I collagen, indicating the presence of post-hypertrophic differentiated neoplastic chondrocytes. Chondrocytes exhibiting a range of phenotypes were found to be present in the same neoplasm. The different observed phenotypes, including the dedifferentiated phenotype, were in contrast to the anaplastic cells of high-grade chondrosarcomas. Comparison of expression data with tumor morphology revealed a relationship between the cellular phenotypes, the tumor matrix composition, and the matrix and cell morphology within the neoplasms. The distinctly different phenotypes of neoplastic chondrocytes are the basis of the characteristic high biochemical and morphological heterogeneity of chondroid neoplasms and shed light on their biological and clinical behavior.     

The effect of hyaluronic acid with different molecular weights on collagen crosslink synthesis in cultured chondrocytes embedded in collagen gels

Hyaluronic acid (HA) is a component of the extracellular matrix of cartilage and has various effects on three-dimensional cultured chondrocytes. We measured Pyridinoline (Pyr), which is a crosslink of collagen in cultured chondrocyte-collagen composites treated with HA of different molecular weights to investigate the effects of the various molecular weights on collagen crosslink synthesis. The control group was collagen gel without cells; group N was treated without HA; and the others were treated with HA with an average molecular weight of 2.3 x10(6) Da (group H), 8.0 x10(5) Da (group M), and 2.3 x10(4) Da (group L). In the control group, the Pyr content decreased, at week 4, being one-tenth that of preculture levels. In groups H and M, it was significantly greater than that in groups L and N at week 4. Pyr/hydroxyproline, which indicates the concentration of Pyr per collagen, decreased greatly in the control group at week 3. In groups H and M, it was significantly higher than that in groups L and N at week 4 and increased to 80 and 76% of normal rabbit articular cartilage, respectively. The concentration of Pyr per collagen in cultured chondrocyte-collagen composites was similar to that of normal articular cartilage in vivo, and higher molecular weight HA may have a greater effect on the maturation of collagen in the composite.     

Modulation of the proliferation and matrix synthesis of chondrocytes by dynamic compression on genipin-crosslinked chitosan/collagen scaffolds

Dynamic compression is an important physical stimulus for the physiology of chondrocyte and articular cartilage tissue engineering. In this study, modulation of chondrocyte behaviors in chitosan/collagen scaffolds with different mechanical properties under free-swelling or dynamic compression conditions was investigated. Rabbit chondrocytes were seeded in chitosan/collagen scaffolds crosslinked by genipin (GP) with different concentrations, and then cultured for 3?days prior to cyclic compression of 40% strain, 0.1?Hz, and 30?min/day for 2?weeks. The results showed that the cell proliferation was increased with increasing genipin concentrations and dynamic compression. On the other hand, although total glycosaminoglycans (GAGs) deposition was enhanced by dynamic compression under certain conditions, e.g. the GP0.5 chitosan/collagen scaffolds for 1?week of compression culture, normalized GAGs deposition per cell was decreased by dynamic compression. Our results suggest that while several studies suggest that dynamic compression benefits articular cartilage tissue engineering, many factors including scaffold types and compression conditions determine the outcome of dynamic compression culture.     

Extracellular matrix protein gene expression of bovine chondrocytes cultured on resorbable scaffolds

It has been demonstrated that using cultured chondrocytes that have been seeded onto various biomatrices can enhance the quality of the articular cartilage repair tissue. As tissue-engineering becomes increasingly more complex there is a need to understand how a specific biomaterial may influence gene expression. In this study several commonly used scaffold materials for cartilage tissue engineering were evaluated with respect to their influence on matrix gene expression. Primary cultures of bovine chondrocytes were established in monolayer then seeded onto polylactic acid (PLLA), polyglycolic acid (PGA), collagen matrices. The induction of collagen type I, collagen type II, and aggrecan was observed at various time points on these biomaterials using RT-PCR. The collagen type I gene was upregulated on collagen scaffolds throughout the culture period. PLLA and PGA showed initial induction followed by downregulation. Monolayer culture did not induce collagen I message. Collagen II genes were selectively upregulated after 72 and 96 h post seeding depending the scaffold material. Monolayer culture had strong induction of collagen II. The aggrecan protein was consistently expressed in all scaffold materials cultures and monolayer.     

Modulation of gene expression of rabbit chondrocytes by dynamic compression in polyurethane scaffolds with collagen gel encapsulation

Chondrocytes have been demonstrated to be sensitive to mechanical stimuli, such as compression, tension, shear force, and hydrostatic pressure. The responses of chondrocytes to mechanical compression have been often studied in vitro with cartilage and chondrocyte/hydrogel systems. The aim of this study was to investigate the effects of dynamic compression on gene expression of rabbit chondrocytes which were seeded in elastic polyurethane scaffolds with or without collagen gel encapsulation. Dynamic compression of 20% or 30% strain with 0.1 Hz frequency was applied to the cell-seeded scaffolds for 4, 8, 12, or 24 h, and then the expression of the three genes related to chondrogenic phenotype, type I and II collagens and aggrecan, was analyzed by RT-PCR. We also investigated the gene expression of the compressed chondrocytes, which had experienced 12-h 30% strain dynamic loading, during the post-compression resting period. We found that the expression of type II collagen did not seem to respond to cyclic compression. On the other hand, aggrecan gene was stimulated by dynamic compression. The stimulatory effect disappeared gradually after the dynamic compression was ceased. Furthermore, the mechano-response of the chondrocytes to aggrecan expression was delayed by collagen gel encapsulation. The expression of type I collagen was enhanced by collagen gel. We found that collagen gel encapsulation prolonged the expression of aggrecan and type I collagen during post-compression resting period. We demonstrated that mechanical and biochemical stimuli modulate the gene expression of chondrocytes.     

The modulation of integrin expression by the extracellular matrix in articular chondrocytes

Normal articular cartilage is composed of chondrocytes embedded within an extracellular matrix (ECM). The patterns of integrin expression determine the adhesive properties of cells by modulating interactions with specific ECMs. Our hypothesis is that chondrocyte integrin expression changes in response to changes in their microenvironment. Porcine articular chondrocytes were encapsulated in alginate beads with several ECMs (collagen type I, collagen type II and fibronectin) for 7 days, subjected to RT-PCR, western blot analysis and immunofluorescence staining. It was found that chondrocytes in different ECMs showed different patterns of integrin expression. Integrin alpha5 and beta1 were strongly expressed in all groups, but integrin alpha1 was strongly expressed only in collagen type I and fibronectin conjugated alginate beads, and integrin alpha2 was strongly expressed only in collagen type II conjugated alginate beads. These findings suggest that the addition of different ECMs to chondrocytes can modulate the patterns and levels of integrin expression possibly through a feedback mechanism. These finding suggest that the modulation of ECM interactions may play a critical role in the pathogenesis of osteoarthritis.     

京尼平苷干预体外培养大鼠软骨细胞Ⅱ型胶原蛋白的合成

背景：近年来的研究表明,中药栀子可能具有活化软骨细胞的功能,而京尼平苷是栀子中重要成分之一。 目的：观察京尼平苷对体外培养大鼠膝关节软骨细胞Ⅱ型胶原蛋白合成的影响。 方法：体外分离培养大鼠膝关节软骨细胞,分别应用25,50及100 mmol/L京尼平苷干预,并设置正常对照组。RT-PCR和Western blot法观察京尼平苷对各组细胞Ⅱ型胶原mRNA和蛋白表达的影响。 结果与结论：RT-PCR和Western blot法检测结果显示,与正常对照组相比,25,50及100 mmol/L京尼平苷干预可以明显促进Ⅱ型胶原mRNA及蛋白的表达(P < 0.01)。结果证实,京尼平苷可以促进大鼠膝关节软骨细胞Ⅱ型胶原蛋白的合成。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

Encapsulating chondrocytes in copolymer gels: bimodal degradation kinetics influence cell phenotype and extracellular matrix development

Hydrogels provide an ideal environment for encapsulating chondrocytes and facilitating the production of cartilaginous tissue. However, the deposition of extracellular matrix (ECM) and ultimate tissue function are significantly affected by degradation of gel scaffolds. It was hypothesized that a bimodal degradation process would capture the critical features necessary for neotissue development. Specifically, most of the initial crosslinks would degrade quickly and enable ECM deposition, whereas a critical amount would remain or degrade much more slowly to provide structural integrity over a longer time period. In this study, chondrocytes were encapsulated in copolymer gels of nondegradable [poly(ethylene glycol) dimethacrylate] and degradable [poly(lactic acid)-b-poly(ethylene glycol)-b-poly(lactic acid) dimethacrylate] macromers to investigate the effects of gel degradation on ECM evolution. All gels were synthesized from 10 wt % total macromer solutions consisting of 0, 19, 21, 23, 25, or 100 mol % nondegradable units. The copolymer constructs were found to have lower DNA content than completely degradable constructs after 8 weeks. However, total biochemical content was very similar among the various copolymer constructs. Histological analysis gave more interesting insight, showing a more uniform spatial distribution of ECM components in copolymer samples than in constructs with 100 mol % nondegradable units. In addition, a number of major structural defects were present in constructs with 0 mol % nondegradable units that became less apparent as the amount of nondegradable units was increased. Overall, the copolymer gels had a higher compressive modulus during neotissue development and also showed no evidence of chondrocyte dedifferentiation. With their bimodal degradation profile, copolymer gels with carefully selected ratios of degrading to slow or nondegrading crosslinks provide distinct advantages for ECM development in tissue-engineered cartilage.     

Activin alters the kinetics of endoderm induction in embryonic stem cells cultured on collagen gels

Embryonic stem cell-derived endoderm is critical for the development of cellular therapies for the treatment of disease such as diabetes, liver cirrhosis, or pulmonary emphysema. Here, we describe a novel approach to induce endoderm from mouse embryonic stem (mES) cells using fibronectin-coated collagen gels. This technique results in a homogeneous endoderm-like cell population, demonstrating endoderm-specific gene and protein expression, which remains committed following in vivo transplantation. In this system, activin, normally an endoderm inducer, caused an 80% decrease in the Foxa2-positive endoderm fraction, whereas follistatin increased the Foxa2-positive endoderm fraction to 78%. Our work suggests that activin delays the induction of endoderm through its transient precursors, the epiblast and mesendoderm. Long-term differentiation displays a twofold reduction in hepatic gene expression and threefold reduction in hepatic protein expression of activin-treated cells compared with follistatin-treated cells. Moreover, subcutaneous transplantation of activin-treated cells in a syngeneic mouse generated a heterogeneous teratoma-like mass, suggesting that these were a more primitive population. In contrast, follistatin-treated cells resulted in an encapsulated epithelial-like mass, suggesting that these cells remained committed to the endoderm lineage. In conclusion, we demonstrate a novel technique to induce the direct differentiation of endoderm from mES cells without cell sorting. In addition, our work suggests a new role for activin in induction of the precursors to endoderm and a new endoderm-enrichment technique using follistatin.     

Immunolocalization of extracellular matrix proteins and collagen synthesis in first-trimester human decidua

The extracellular matrix (ECM) of first-trimester human decidua was examined with indirect immunofluorescence using affinity-purified antibodies to human collagen types I, III, IV, V, laminin, and fibronectin. In addition, the validity of the classification "mesenchymal-epithelioid" for differentiated decidual cells was addressed using antibodies to the intermediate filament proteins, vimentin, a mesenchymal marker, and keratin, an epithelial marker. Biosynthesis of extracellular matrix components was examined by radiolabeling of decidual explants in culture with 3H-proline, followed by immunoprecipitations of synthesized proteins with collagen type-specific antibodies. Immunofluorescence showed decidual cells are embedded in an extensive network of collagen types I and III, and intracytoplasmic staining suggested synthesis of these collagens by the decidual cells. Collagen type IV and laminin localized in the external lamina which surrounds the differentiated decidual cell, and some fluorescence was evident in the peripheral cytoplasm. Immunoreactive collagen type V was observed in close association with the external lamina and in the peridecidual matrix. Fibronectin localized throughout the decidual ECM and in fibrillar and punctuate patterns in the decidual cell cytoplasm. Differentiated decidual cells retained a "mesenchymal" intermediate filament cytoskeleton containing an abundance of vimentin filaments, but very few, if any, keratin filaments. Collagen types I, III, V, and to a lesser extent, IV, were immunoprecipitated from the medium of decidual explants after 24 hours of culture, demonstrating in vitro synthesis and secretion of these collagens by first trimester human decidua.     

Differences between sub-populations of cultured bovine articular chondrocytes. I. Morphology and cartilage matrix production

Bovine articular chondrocytes cultured in agarose gel comprise a heterogeneous population when judged by morphological and histochemical criteria. The purpose of the present experiments was to compare, under the same conditions of culture, sub-populations of chondrocytes derived from different depths of articular cartilage. Sub-populations of chondrocytes were cultured separately following their isolation from slices of articular cartilage cut from successive depths of the tissue. Chondrocytes derived from superficial and deep zones differed significantly in morphology, rate of proliferation, and activity in secreting a proteoglycan-rich extracellular matrix. The differences are sufficient to account for the heterogeneity observed in cultures of the entire cell population, and the correlate well with known variations with depth in morphology and histochemistry of intact articular cartilage. These results demonstrate that articular chondrocytes continue in culture to express metabolic differences which reflect their original anatomical location; such differences may have important functional significance.     

Modulation of extracellular matrix metabolism in rabbit articular chondrocytes and human rheumatoid synovial cells by the non-steroidal anti-inflammatory drug etodolac. I: Collagen synthesis

Cultures of human rheumatoid synovial cells and rabbit articular chondrocytes were exposed to various concentrations of Etodolac (from 0.01 to 10 g/ml) in presence or absence of 500 pg/ml (5 U/ml) human recombinant Interleukin-1 (IL-1). Incubation of chondrocytes with Etodolac for 24 h did not alter collagen biosynthesis. In contrast, 1 g/ml Etodolac caused a 20% increase of collagen production in synoviocytes. Addition of Etodolac in combination with IL-1 could partially suppress the inhibitory effect exerted by the cytokine on both cell types. Four-day exposure of chondrocytes to 0.1 and 1 g/ml Etodolac led to an increased accumulation of collagen in the cell layer compartment. However, this treatment could not prevent the inhibitory effect of IL-1 on this collagen fraction. Treatment of synoviocytes for eight days with the same concentrations of Etodolac did not modify their collagen production but suppressed totally the inhibitory effect of IL-1. These data show that Etodolac is able to augment chondrocyte metabolism during a long term treatment. Moreover, under certain conditions, this drug can reduce or even suppress the IL-1-induced inhibition of collagen biosynthesis, a process that may take a part in the connective tissue alterations associated with osteoarticular diseases such as rheumatoid arthritis and osteoarthritis.We thank R. Béliard (Laboratoire d'Anatomie Pathlogique, C.H.U. Caen) for expert technical assistance and Dr. D. Lando (Roussel Uclaf, France) for kindly providing human recombinant Interleukin-1. This work was financially supported by Wyeth France.     

Maintenance of cartilaginous gene expression on extracellular matrix derived from serially passaged chondrocytes during in vitro chondrocyte expansion

The loss of cartilaginous phenotype during in vitro expansion culture of chondrocytes is a major barrier for the application of cartilage tissue engineering. The use of matrices mimicking the in vivo extracellular matrix (ECM) microenvironment is anticipated to be an efficient method to suppress chondrocyte phenotype loss. In this study, we developed several types of ECM derived from serially passaged chondrocytes for use as cell-culture substrata and compared their effects on chondrocyte functions. Primary bovine chondrocytes and serially passaged chondrocytes (at passages 2 and 6) were cultured on tissue-culture polystyrene. After culture, the cellular components were selectively removed from the ECM deposited by the cells. The remaining ECM proteins were used as cell-culture substrata. The composition of the deposited ECM depended on the culture stage of the serially passaged chondrocytes used for the ECM production. The deposited ECM supported the adhesion and proliferation of chondrocytes. The effects of the ECM on the chondrocyte dedifferentiation during in vitro passage culture differed dramatically depending on the phenotype of the chondrocytes used to produce the ECM. The primary chondrocyte-derived ECM delayed the chondrocyte dedifferentiation during in vitro passage culture and is a good candidate for chondrocyte subculture for tissue engineering.     

Formation of extracellular matrix by cultured rat mesangial cells.

Formation of extracellular matrix (ECM) by mesangial cells (MCs) contributes to progressive glomerulosclerosis. The authors investigated the production and distribution of ECM constituents by cultured rat MCs, using immunocytochemistry and immunoelectron microscopy. Staining for all ECM constituents increased after serum feeding. Localization was strictly intracellular until confluency, when extracellular deposition of collagen IV and laminin appeared, followed by fibronectin and collagen III. In parallel, the intracellular staining for these proteins diminished markedly. Neither extracellular deposition nor intracellular loss was observed for collagen I and thrombospondin. On surfaces coated with collagen IV or laminin, extracellular deposition of ECM constituents clearly preceded confluency. These results indicate that synthesis of ECM constituents parallels MC growth, and that extracellular deposition of ECM occurs at cell-cell contact. Collagen IV or laminin secreted by MCs in the substratum accelerates production and facilitates secretion of other ECM constituents in an autocrine fashion.     

Histamine alters gene expression in cultured human nasal epithelial cells

BACKGROUND: Airway epithelial cells produce cytokines and participate in the regulation of mucosal immunity. Although nasal epithelial cells express histamine receptors, it is not exactly known how nasal epithelial cells respond to histamine. OBJECTIVE: The objective of this study was to examine whether histamine can alter the expression of the 4 genes encoding H1 receptor, IL-8, TNF-alpha, and ZO-1 tight-junction protein in cultured nasal epithelial cells. METHODS: We added histamine or vehicle to cultured human nasal epithelial cells and extracted RNA from them 4 hours later. After DNase treatment, mRNAs of beta-actin, H1 receptor, IL-8, TNF-alpha, and ZO-1 tight-junction protein were amplified by using RT-PCR. RESULTS: Histamine significantly upregulated IL-8 mRNA expression and significantly downregulated ZO-1 mRNA expression. The latter effect was blocked by pretreatment with mepyramine, an H1 receptor antagonist. CONCLUSION: The reduction of ZO-1 mRNA by histamine may cause increased permeability of the mucosa during allergic reactions in the nose.     

Synthesis and supramolecular organization of components of the extracellular matrix by fibroblasts cultured in a collagen lattice]

Fibroblasts cultured in a collagen gel contract and organize the gel into a three-dimensional matrix of collagen fibers. Within this matrix, the fibroblast cell cycle is blocked at the G1 phase but also at the G2 phase. The fibroblasts produce the main extracellular matrix components (collagen, noncollagen proteins, glycosaminoglycans), although in small amounts. Studies using this in vitro model with radiolabeled precursor substances (14C proline, 3H glucosamine) demonstrated production of supermolecular complexes which resisted to proteolysis by pepsin and collagenase and could not be isolated by saline precipitation. Polyclonal antibodies identified type I collagen, type VI collagen and fibronectin in this coherent supermolecular structure. The presence of glycosaminoglycans was also demonstrated by alcian blue precipitation.     

Selective progesterone receptor modulator asoprisnil down-regulates collagen synthesis in cultured human uterine leiomyoma cells through up-regulating extracellular matrix metalloproteinase inducer

BACKGROUND: A recent clinical trial demonstrated that selective progesterone receptor modulator asoprisnil is effective in reducing uterine leiomyoma volume. We investigated the effects of asoprisnil in vitro on the expression of the extracellular matrix (ECM)-remodeling enzymes and collagens in cultured leiomyoma and matching normal myometrial cells. METHODS: The expression of extracellular matrix metalloproteinase inducer (EMMPRIN), matrix metalloproteinases (MMPs), tissue inhibitors of MMP (TIMPs) and collagens were assessed by western blot analysis. RESULTS: Untreated cultured leiomyoma cells had significantly lower EMMPRIN (P < 0.05), MMP-1 (P < 0.05) and membrane type 1-MMP (MT1-MMP) (P < 0.01) protein contents, but significantly higher TIMP-1 (P < 0.05), TIMP-2 (P < 0.01), type I (P < 0.05) and type III (P < 0.01) collagen protein contents compared with untreated cultured myometrial cells. Treatment with asoprisnil at concentrations > or =10(-7) M for 48 h significantly (P < 0.05) increased EMMPRIN, MMP-1 and MT1-MMP protein contents, and decreased TIMP-1 (P < 0.05), TIMP-2 (P < 0.01), type I (P < 0.01) and type III (P < 0.05 at 10(-7) M; P < 0.01 at 10(-6) M) collagen protein contents in cultured leiomyoma cells compared with control cultures. However, asoprisnil treatment did not affect the protein contents of ECM-remodeling enzymes and collagens in cultured myometrial cells. CONCLUSIONS: These results suggest that asoprisnil may reduce collagen deposit in the ECM of cultured leiomyoma cells through decreasing collagen synthesis and enhancing the expression of EMMPRIN, MMPs and TIMPs without comparable effects on cultured myometrial cells.