Chondrogenic potential of skeletal cell populations: selective growth of chondrocytes and their morphogenesis and development in vitro

Most vertebrate embryonic and post-embryonic skeletal tissue formation occurs through the endochondral process in which cartilage serves a transitory role as the anlage for the bone structure. The differentiation of chondrocytes during this process in vivo is characterized by progressive morphological changes associated with the hypertrophy of these cells and is defined by biochemical changes that result in the mineralization of the extracellular matrix. The mechanisms, which, like those in vivo, promote both chondrogenesis in presumptive skeletal cell populations and endochondral progression of chondrogenic cells, may be examined in vitro. The work presented here describes mechanisms by which cells within presumptive skeletal cell populations become restricted to a chondrogenic lineage as studied within cell populations derived from 12-day-old chicken embryo calvarial tissue. It is found that a major factor associated with selection of chondrogenic cells is the elimination of growth within serum-containing medium. Chondrogenesis within these cell populations appears to be the result of permissive conditions which select for chondrogenic proliferation over osteogenic cell proliferation. Data suggest that chondrocyte cultures produce autocrine factors that promote their own survival or proliferation. The conditions for promoting cell growth, hypertrophy, and extracellular matrix mineralization of embryonic chicken chondrocytes in vitro include ascorbic acid supplementation and the presence of an organic phosphate source. The differentiation of hypertrophic chondrocytes in vitro is associated with a 10-15-fold increase in alkaline phosphatase enzyme activity and deposition of mineral within the extracellular matrix. Temporal studies of the biochemical changes coincident with development of hypertrophy in vitro demonstrate that proteoglycan synthesis decreases 4-fold whereas type X collagen synthesis increases 10-fold within the same period. Ultrastructural examination reveals cellular and extracellular morphology similar to that of hypertrophic cells in vivo with chondrocytes embedded in a well formed extracellular matrix of randomly distributed collagen fibrils and proteoglycan. Mineral deposition is seen in the interterritorial regions of the matrix between the cells and is apatitic in nature. These characteristics of chondrogenic growth and development are very similar in vivo and in vitro and they suggest that studies of chondrogenesis in vitro may provide a valuable model for the process in vivo.     

Determination of substance P in human nasal lavage fluid

Depending on the cell type studied, the involvement of type II transglutaminase (TGase) has been proposed in almost any event of the cell life such as differentiation, apoptosis, growth, aging, cell morphology and adhesion, metastatic capacity or extracellular matrix stabilization. In order to define the field(s) where this enzyme may be implicated in chondrocytes, type II TGase expression was studied in chondrocytes at different passages which differentiated state was modulated by retinoic acid, dihydrocytochalasin B or staurosporin. Results showed that (i) type II TGase expression is not incompatible with type II collagen expression, a main marker of chondrocyte differentiation (ii) type II TGase expression is higher when cells are in the exponential phase of growth than when growth arrested (iii) a high type II TGase expression does not imply that cells are apoptotic although cell apoptosis correlates with increased type II TGase expression (iv) non-adherent cells do not express type II TGase whereas adherent cells do whatever their differentiation state as assessed by type II collagen synthesis. These results suggest that, in articular chondrocytes, type II TGase is specifically implicated in the cell adhesion capacity.     

The conformational equilibrium of human growth hormone

To examine the role of bone morphogenetic protein (BMP) signaling in chondrocytes during endochondral ossification, the dominant negative (DN) forms of BMP receptors were introduced into immature and mature chondrocytes isolated from lower and upper portions of chick embryo sternum, respectively. We found that control sternal chondrocyte populations expressed type IA, IB, and II BMP receptors as well as BMP-4 and -7. Expression of a DN-type II BMP receptor (termed DN-BMPR-II) in immature lower sternal (LS) chondrocytes led to a loss of differentiated functions; compared with control cells, the DN-BMPR- II-expressing LS chondrocytes proliferated more rapidly, acquired a fibroblastic morphology, showed little expression of type II collagen and aggrecan genes, and upregulated type I collagen gene expression. Expression of DN-BMPR-II in mature hypertrophic upper sternal (US) chondrocytes caused similar effects. In addition, the DN-BMPR-II-expressing US cells exhibited little alkaline phosphatase activity and type X collagen gene expression, while the control US cells produced both alkaline phosphatase and type X collagen. Both DN-BMPR-II-expressing US and LS chondrocytes failed to respond to treatment with BMP-2 . When we examined the effects of DN forms of types IA and IB BMP receptors, we found that DN-BMPR-IA had little effect, while DN-BMPR-IB had similar but weaker effects compared with those of DN-BMPR-II. We conclude that BMP signaling, particularly that mediated by the type II BMP receptor, is required for maintenance of the differentiated phenotype, control of cell proliferation, and expression of hypertrophic phenotype.     

Determinants of tPA antigen and associations with coronary artery disease and acute cerebrovascular disease

A serum-free organ culture model for chondrocyte maturation, using the Avian sternum, was developed. Day-14 chick embryo sterna were placed in organ culture in the presence of defined medium. The optimal medium for chondrocyte terminal differentiation contained specific concentrations of dexamethasone, insulin, thyroid hormone and ascorbic acid. Three parameters, including sternal growth, cell diameter and type X collagen production, were analyzed as indicators of chondrocyte terminal differentiation. These parameters were analyzed in cephalic, middle and caudal regions of the organ-cultured chick sterna and compared to sterna grown in ovo. This study demonstrates that the organ-cultured tissue maintains normal morphological characteristics and terminal differentiation in the cephalic region only, similar to in ovo development, while maintaining normal cell-matrix relationships.     

Inhibition of proteoglycan biosynthesis decreases the calcification of chondrocyte cultures

In order to determine the role of proteoglycan in the calcification of cartilage, the effects on calcifying chondrocyte cultures of treatments that disrupt proteoglycan biosynthesis have been studied. Treatment of secondary cultures of embryonic chick chondrocytes with non-toxic concentrations of the beta-xyloside p-nitrophenyl beta-D-xylopyranoside (PNPX) resulted in dose-dependent inhibition of both proteoglycan and mineral deposition. Based on the expression of Type X collagen, however, PNPX is also a potent inhibitor of chondrocyte differentiation. Under-sulfation of proteoglycans was effected by growth of chondrocyte cultures in sulfate-depleted medium. Growth in low-sulfate medium did not significantly affect the growth or differentiation of these cultures, but caused an approximate two-fold decrease in mineral content compared to cultures grown in normal medium. These findings indicate that disruption of proteoglycan biosynthesis in chondrocyte cultures results in decreased levels of calcification. Therefore, proteoglycans appear to function as promoters of chondrocyte calcification.     

Genetic and clinical features of sensorineural hearing loss associated with the 1555 mitochondrial mutation

This article describes the modulation, by extracellular collagen, of DNA and proteoglycan synthesis in articular chondrocytes stimulated with transforming growth factor-beta 1. Type-I and type-II collagen, heat-denatured type-II collagen, and bovine serum albumin were each incorporated into alginate in increasing concentrations. Bovine articular chondrocytes were isolated and were resuspended in the alginate, yielding alginate beads with final extracellular protein concentrations of 0-1.5% (wt/vol) for the collagens and 0-2.5% (wt/vol) for bovine serum albumin. Cultures of beads were maintained for 7 days in basal Dulbecco's modified Eagle medium or in medium supplemented with 10 ng/ml transforming growth factor-beta 1. Subsequently, the synthesis of DNA and proteoglycan was measured by radiolabel-incorporation methods with [35S]sulfate and [3H]thymidine, and the values were normalized to the DNA content. Transforming growth factor-beta 1 stimulated the synthesis of both DNA and proteoglycan in a bimodal fashion. The presence of extracellular type-II collagen increased the rate of DNA and proteoglycan synthesis in a dose-dependent fashion in cultures stimulated by transforming growth factor-beta 1, whereas heat-inactivated type-II collagen abrogated the effects observed with type-II collagen for synthesis of both DNA and proteoglycan. In contrast, the presence of extracellular type-I collagen caused a dose-dependent inhibition of synthesis of both DNA and proteoglycan in cultures stimulated with transforming growth factor-beta 1. Extracellular bovine serum albumin brought about a limited increase in synthesis rates, presumably by blocking nonspecific cytokine binding. These results suggest that type-II collagen has a specific role in chondrocyte regulation and serves to mediate the response of chondrocytes to transforming growth factor-beta 1.     

The role of 12-lipoxygenase in pancreatic -cells (Review)

A culture system that facilitates the chondrogenic differentiation of rabbit bone marrow-derived mesenchymal progenitor cells has been developed. Cells obtained in bone marrow aspirates were first isolated by monolayer culture and then transferred into tubes and allowed to form three-dimensional aggregates in a chemically defined medium. The inclusion of 10(-7) M dexamethasone in the medium induced chondrogenic differentiation of cells within the aggregate as evidenced by the appearance of toluidine blue metachromasia and the immunohistochemical detection of type II collagen as early as 7 days after beginning three-dimensional culture. After 21 days, the matrix of the entire aggregate contained type II collagen. By 14 days of culture, there was also evidence for type X collagen present in the matrix and the cells morphologically resembled hypertrophic chondrocytes. However, chondrogenic differentiation was achieved in only approximately 25% of the marrow cell preparations used. In contrast, with the addition of transforming growth factor-beta 1 (TGF-beta 1), chondrogenesis was induced in all marrow cell preparations, with or without the presence of 10(-7) M dexamethasone. The induction of chondrogenesis was accompanied by an increase in the alkaline phosphatase activity of the aggregated cells. The results of RT-PCR experiments indicated that both type IIA and IIB collagen mRNAs were detected by 7 days postaggregation as was mRNA for type X collagen. Conversely, the expression of the type I collagen mRNA was detected in the preaggregate cells but was no longer detectable at 7 days after aggregation. These results provide histological, immunohistochemical, and molecular evidence for the in vitro chondrogenic differentiation of adult mammalian progenitor cells derived from bone marrow.     

Collagen type I modulates the platelet-derived growth factor (PDGF) regulation of the growth and expression of beta1 integrins by rat mesangial cells

Mesangial cell (MC) proliferation and the deposition of collagen type I (collagen I) are the major pathological features in many types of glomerulonephritis (GN). Recent work suggested that beta-integrins play a critical role in the cell proliferation and extracellular matrix (ECM) remodeling observed in tissue repair after injury. To examine the involvement of beta-integrins in MC proliferation in association with the interaction of MCs with pathological collagen I, we investigated the effect of a prominent mitogen, platelet-derived growth factor-BB (PDGF-BB) on the growth and expression of beta-integrins by MCs cultured on plastic or in a three-dimensional collagen I gel. Immunoprecipitation using 35S-metabolic labeling, flow cytometry and a 3H-thymidine-uptake analysis demonstrated that PDGF-BB stimulated the cell mitogenicity and the expression of alpha5beta1 integrin (a fibronectin receptor), but not alpha1beta1 integrin (a collagen and laminin receptor) of MCs on plastic, in a dose-dependent manner. In contrast, MCs in the collagen I gels showed no significant changes in mitogenicity or alpha1beta1 and alpha5beta1 integrin expression, but increased alpha1beta1 integrin-mediated gel contraction was observed after PDGF-BB stimulation. Thus, the parallel up-regulation of MC-mitogenicity and alpha5beta1 integrin expression by PDGF-BB suggested that alpha5beta1 integrin is an important ECM receptor involved in the proliferative phenotype of MC. A spatial interaction between MCs and pathological collagen I in GN may influence the PDGF regulation of the MC phenotype regarding the cell growth and the expression of beta1 integrins.     

Inhibition of glomerular mesangial cell proliferation and extracellular matrix deposition by halofuginone

Mesangial cell proliferation, increased deposition of collagen, and expansion of the mesangial extracellular matrix (ECM) are key features in the development of mesangioproliferative diseases. Halofuginone, a low molecular weight anti-coccidial quinoazolinone derivative, inhibits collagen type alpha 1(I) gene expression and synthesis. We investigated the effect of halofuginone on both normal and SV40 transformed mesangial cell proliferation, collagen synthesis, and ECM deposition. Proliferation of both cell types was almost completely inhibited in the presence of 50 ng/ml halofuginone. The cells were arrested in the late G1 phase of the cell cycle and resumed their normal growth rate following removal of the compound from the culture medium. The antiproliferative effect of halofuginone was associated with inhibition of tyrosine phosphorylation of cellular proteins. Similar results were obtained whether the mesangial cells were seeded on regular tissue culture plastic or in close contact with a naturally produced ECM resembling their local environment in vivo. Halofuginone also inhibited synthesis and deposition of ECM by mesangial cells as indicated by a substantial reduction in 14C-glycine and Na2(35)SO4 incorporation into the ECM, and by the inhibition of collagen type I synthesis and gene expression. It is proposed that by inhibiting collagen type I synthesis and matrix deposition, halofuginone exerts a potent antiproliferative effect that may be applied to inhibit mesangial cell proliferation and matrix expansion in a variety of chronic progressive glomerular diseases.     

Human chondrocytes proliferate and produce matrix components in microcarrier suspension culture

Chondrocytes propagated in monolayer culture proliferate and change into 'fibroblastoid'-like cells. This change is characterized by a shift in production of collagen type II to I and from high- to low-molecular-weight proteoglycans. When propagated in three-dimensional culture, chondrocytes have limited ability to divide but re-express their original characteristics. The goal of the present study was to determine whether a microcarrier suspension culture system would support chondrocyte proliferation and phenotype expression. Our experiments indicate that a collagen type I microcarrier (cellagen) best supported chondrocyte proliferation and phenotype expression. Cells in cellagen microcarriers multiplied at least twentyfold within 2 weeks and had doubling times of 2 to 3 d. Viable and metabolically active cells were retrieved with ease. The harvested chondrocytes had no detectable staining for collagen type I and stained intensely for collagen type II. Our studies demonstrate that the microcarrier suspension culture system supports growth and enhances expression of the 'chondrocytic' phenotype. Attachment to a constrained surface and the fluid shear forces on the microcarriers during suspension culture may have helped chondrocytes to reacquire their rounded shape and produce cartilage matrix components.     

Culture of chondrocytes in alginate and collagen carrier gels

In this in vitro study, we compared the potential of collagen and alginate gels as carriers for chondrocyte transplantation and we studied the influence of demineralized bone matrix (DBM) on chondrocytes in the gels. Chondrocytes were assessed for cell viability, phenotype (histology), proliferation rate and sulfate incorporation. Collagen gels showed a significant increase in cell numbers, but the chondrocytes dedifferentiated into fibroblast-like cells from day 6 onwards. In alginate gels, initial cell loss was found, but the cells maintained their typical chondrocyte phenotype. Although the total quantity of proteoglycans initially synthesized per cell in collagen gel was significantly higher, expressed per cell, the quantity in alginate gel eventually surpassed collagen. No effects of culturing chondrocytes in combination with DBM could be demonstrated on cell proliferation and sulfate incorporation. The collagen and alginate gels have different advantages as carriers for chondrocyte transplantation. The high proliferation rate of chondrocytes in collagen gel may be an advantage, but the preservation of the chondrocyte phenotype and the gradually increasing proteoglycan synthesis in alginate gel is a promising method for creating a hyaline cartilage implant in vitro.     

Integrin alpha 2 beta 1 is a positive regulator of collagenase (MMP-1) and collagen alpha 1(I) gene expression

A classical model for studying the effects of extracellular matrix is to culture cells inside a three-dimensional collagen gel. When surrounded by fibrillar collagen, many cell types decrease the production of type I collagen, and the expression of interstitial collagenase (matrix metalloproteinase-1; MMP-1) is simultaneously induced. To study the role of the collagen-binding integrins alpha 1 beta 1 and alpha 2 beta 1 in this process, we used three different osteogenic cell lines with distinct patterns of putative collagen receptors: HOS cells, which express only alpha 1 beta 1 integrin, MG-63 cells, which express only alpha 2 beta 1 integrin, and KHOS-240 cells, which express both. Inside collagen gels, alpha 1 (I) collagen mRNA levels were decreased in HOS and KHOS-240 cells but not in MG-63 cells. In contrast, MMP-1 expression was induced in KHOS-240 and MG-63 cells but not in HOS cells. Transfection of MG-63 cells with alpha 2 integrin cDNA in an antisense orientation reduced the expression level of alpha 2 integrin. These cell clones showed induction and reduction of mRNA levels for MMP-1, respectively. HOS cells normally lacking alpha 2 beta 1 integrin were forced to express it, and this prevented the down-regulation in the levels of alpha 1 (I) collagen mRNA when cells were grown inside collagen gels. The data indicate that the level of MMP-1 expression is regulated by the collagen receptor alpha 2 beta 1 integrin. The down-regulation of collagen alpha 1 (I) is mediated by another receptor. Integrin alpha 2 beta 1 may compete with it and thus be a positive regulator of collagen synthesis.     

Recombinant bone morphogenetic protein (BMP)-2 regulates costochondral growth plate chondrocytes and induces expression of BMP-2 and BMP-4 in a cell maturation-dependent manner

This study examined the effect of recombinant human bone morphogenetic protein-2 on several parameters of growth, differentiation, and matrix synthesis and on the endogenous production of mRNA of bone morphogenetic proteins 2 and 4 by growth plate chondrocytes in culture. Chondrocytes from resting and growth zones were obtained from rat costochondral cartilage and cultured for 24 or 48 hours in medium containing 0.05-100 ng/ml recombinant human bone morphogenetic protein-2 and 10% fetal bovine serum. Incorporation of [3H]thymidine, cell number, alkaline phosphatase specific activity, incorporation of [3H]proline into collagenase-digestible protein and noncollagenase-digestible protein, and incorporation of [35S]sulfate were assayed as indicators of cell proliferation, differentiation, and extracellular matrix synthesis. mRNA levels for bone morphogenetic proteins 2 and 4 were determined by Northern blot analysis. Recombinant human bone morphogenetic protein-2 increased the incorporation of [3H]thymidine by quiescent resting-zone and growth-zone cells in a similar manner, whereas it had a differential effect on nonquiescent cultures. At 24 and 48 hours, 12.5-100 ng/ml recombinant human bone morphogenetic protein-2 caused a dose-dependent increase in cell number and DNA synthesis in resting-zone chondrocytes. No effect was seen in growth-zone cells. Recombinant human bone morphogenetic protein-2 stimulated alkaline phosphatase specific activity in resting-zone chondrocytes in a bimodal manner, causing significant increases between 0.2 and 0.8 ng/ml and again between 25 and 100 ng/ml. In contrast, alkaline phosphatase specific activity in growth-zone chondrocytes was significantly increased only between 12.5 and 100 ng/ml. Recombinant human bone morphogenetic protein-2 increased the production of both collagenase-digestible protein and noncollagenase-digestible protein by resting-zone and growth-zone cells, but incorporation of [35S]sulfate was unaffected. Administration of recombinant human bone morphogenetic protein-2 also increased incorporation of [3H]uridine in both resting-zone and growth-zone chondrocytes; these cells produced mRNA for bone morphogenetic proteins 2 and 4. Bone morphogenetic protein-2 mRNA levels in both resting-zone and growth-zone chondrocytes increased in the presence of recombinant human bone morphogenetic protein-2; however, bone morphogenetic protein-4 mRNA levels in growth-zone cells decreased under its influence, and those in resting-zone cells were upregulated only with a dose of 10 ng/ml. This indicates that recombinant human bone morphogenetic protein-2 regulates chondrocyte proliferation, differentiation, and matrix production, and the effects are dependent on the stage of cell maturation. Resting-zone chondrocytes were more sensitive, suggesting that they are targeted by bone morphogenetic protein-2 and that this growth factor may have autocrine effects on these cells.     

Effect of a combined trenbolone acetate and estradiol implant on steady-state IGF-I mRNA concentrations in the liver of wethers and the longissimus muscle of steers

Endochondral ossification in growth plates proceeds through several consecutive steps of late cartilage differentiation leading to chondrocyte hypertrophy, vascular invasion, and, eventually, to replacement of the tissue by bone. It is well established that the subchondral vascular system is pivotal in the regulation of this process. Cells of subchondral blood vessels act as a source of vascular invasion and, in addition, release factors influencing growth and differentiation of chondrocytes in the avascular growth plate. To elucidate the paracrine contribution of endothelial cells we studied the hypertrophic development of resting chondrocytes from the caudal third of chick embryo sterna in co-culture with endothelial cells. The design of the experiments prevented cell-to-cell contact but allowed paracrine communication between endothelial cells and chondrocytes. Under these conditions, chondrocytes rapidly became hypertrophied in vitro and expressed the stage-specific markers collagen X and alkaline phosphatase. This development also required signaling by thyroid hormone in synergy. Conditioned media could replace the endothelial cells, indicating that diffusible factors mediated this process. By contrast, smooth muscle cells, fibroblasts, or hypertrophic chondrocytes did not secrete this activity, suggesting that the factors were specific for endothelial cells. We conclude that endochondral ossification is under the control of a mutual communication between chondrocytes and endothelial cells. A finely tuned balance between chondrocyte-derived signals repressing cartilage maturation and endothelial signals promoting late differentiation of chondrocytes is essential for normal endochondral ossification during development, growth, and repair of bone. A dysregulation of this balance in permanent joint cartilage also may be responsible for the initiation of pathological cartilage degeneration in joint diseases.     

Terminal differentiation of chondrocytes is arrested at distinct stages identified by their expression repertoire of marker genes

During endochondral bone formation, cells in the emerging cartilaginous model transit through a cascade of several chondrocyte differentiation stages, each characterized by a specific expression repertoire of matrix macromolecules, until, as a final step, the hypertrophic cartilage is replaced by bone. In many permanent cartilage tissues, however, late differentiation of chondrocytes does not occur, due to negative regulation by the environment of the cells. Here, addressing the reason for the difference between chondrocyte fates in the chicken embryo sternum, cells from the caudal and cranial part were cultured separately in serum-free agarose gels with complements defined earlier that either permit or prevent hypertrophic development. Total RNA was extracted using a novel protocol adapted to agarose cultures, and the temporal changes in developmental stage-specific mRNA expression were monitored by Northern hybridization and phosphor image analysis. Kinetic studies of the mRNA accumulation not only showed significant differences between the expression patterns of cranial and caudal cultures after recovery, but also revealed two checkpoints of chondrocyte differentiation in keeping with cartilage development in vivo. Terminal differentiation of caudal chondrocytes is blocked at the late proliferative stage (stage Ib), while the cranial cells can undergo hypertrophic development spontaneously. The differentiation of cranial chondrocytes is reversible, since they can re-assume an early proliferative (stage Ia) phenotype under the influence of insulin, fibroblast growth factor-2 and transforming growth factor-beta in combination. Thus, the expression pattern in the latter culture resembles that of articular chondrocytes. We also provide evidence that the capacities of caudal and sternal chondrocytes to progress from the late proliferative (stage Ib) to hypertrophic stage (stage II) correlate with their differing abilities to express the Indian hedgehog gene.     

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.     

Extracellular matrix in renal cell carcinomas

Extracellular matrix (ECM) may be divided into interstitial matrix and the basement membrane (BM). ECM influences a variety of epithelial cell behaviours, including proliferation, differentiation, and morphogenesis, maybe most widely studied in kidney morphogenesis. In carcinomas, including renal cell carcinomas (RCCs), these properties and interactions of cells with interstitial matrix and BM are disturbed. As a carcinoma with a tendency to spread to distant sites, RCC is an interesting target for the study of epithelial-stromal interactions. Among interstitial collagens, type VI collagen appears to be widely distributed in RCCs. Also EDA-fibronectin (EDA-Fn) as well as tenascin-C (Tn) are important stromal components especially in poorly differentiated carcinomas. BMs of RCC islets and those of tumor blood vessel endothelia may merge in poorly differentiated carcinomas. As a dynamic component of BMs, laminins (Ln) are important in kidney development and RCC progression. Type IV collagen and nidogen, other components of BMs in RCCs, are produced by stromal as well as epithelial cells. ECM proteins may function in RCC progression by binding and regulating the activity of growth factors e.g. transforming growth factor beta 1 and basic fibroblast growth factor. Also the expression of cell surface receptors for ECM is disturbed in RCCs. At least alpha v integrin (Int) and CD44 emerge in renal epithelial cells during malignant transformation. Papillary renal neoplasms differ from RCCs by cell adhesion receptor expression and BM composition as well as by ECM avascularity and capacity to bind growth factors, thus suggesting a distinct property for this renal tumor.     

A conserved helicase motif of the AddA subunit of the Bacillus subtilis ATP-dependent nuclease (AddAB) is essential for DNA repair and recombination

We examined the behavior of fetal rat chondrocytes cultured on a bioactive glass-ceramic containing apatite and wollastonite (A.W.G.C.). Biomaterial surface topography and profiles were evaluated by bidimensional profilometry and revealed a rough surface for the glass-ceramic compared to the plastic coverslips used as controls. Chondrocyte attachment was evaluated by measuring the number of attached cells after one day of culture and by morphological observations. Chondrocytes attached in great numbers to the material surface by means of focal contacts containing vinculin and beta1-integrin. Fluorescent labeling of actin and vimentin revealed a poor spreading of chondrocytes on the bioactive glass-ceramic compared to the plastic coverslips, where the cells appeared to adhere intimately to the surface and exhibited polygonal arrays of stress fibers. During the following days of culture, chondrocytes proliferated, colonized the surface of the material, and, finally, on day 10, formed nodular structures composed of round cells separated by a dense extracellular matrix. Furthermore, these clusters of round cells were positive for type II collagen and chondroitin sulfate, both hard markers of the chondrocyte phenotype. In addition, protein synthesis, alkaline phosphatase activity, and proteoglycan production were found to increase gradually during the culture period with a pattern similar to that observed on control cultures. These results demonstrate that the bioactive glass-ceramic tested in this study appears to be a suitable substrate for in vitro chondrocyte attachment, differentiation, and matrix production.     

Do calcium and zinc ions influence matrix molecule synthesis of chondrocytes?

The experiments described here tested the effect of various calcium (Ca) and Zinc (Zn) concentrations on cell proliferation and matrix molecule synthesis of fetal and adult bovine chondrocytes in monolayer cultures. Levels of Ca < 0.2 mM in a culture medium or the addition of Zn (0.1-50 microM) selectively promoted the production of collagen but did not affect significantly synthesis of proteoglycans. No change in proliferation of fetal and adult chondrocytes could be observed. In contrast 10 mM Ca promoted the hypertrophic differentiation of chondrocytes (e.g. expression of collagen type X). The results are related to calcium channel configurations in chondrocytes in the discussion.