Influence of retinol on human chondrocytes in agarose culture

Vitamin A and its congeners, collectively called retinoids, are known to have teratogenic potential and have induced craniofacial and limb malformations in numerous animal species. More importantly, retinoids are recognized as teratogenic to fetuses of pregnant women who have taken such preparations for dermatologic disorders. Information gathered from the study of animal models suggests that retinoids interfere with cartilage differentiation. If chondrogenesis in limb development is disturbed it may contribute to limb reductions and malformations. In vitro studies using various animal systems have shown that cartilage matrix macromolecules are altered to resemble those secreted by mesenchymal cells. The response of human chondrocytes to retinoids in vitro is not known. Culture of human chondrocytes in agarose maintains the cartilage phenotype and therefore serves as a model system to evaluate the influence of retinoids directly on human chondrogenesis. The studies presented in this paper were done to determine if the expression of specific matrix macromolecules of human chondrocytes in agarose culture is altered by retinol treatment. Immunocytochemistry demonstrated enhanced labeling of type I collagen while type II collagen labeling was reduced in cultures treated with retinol. In addition, morphometric analyses indicated a decrease in the size and number of chondrogenic clusters and that individual cells synthesized less alcian blue matrix when compared to parallel control cultures. The size of the proteoglycan monomers, glycosaminoglycan side chains as well as the disaccharide composition were not affected. However, there was a reduction in the quantity of proteoglycan monomers produced.     

Influence of interleukin-1 on the morphology and proteoglycan metabolism of cultured bovine articular chondrocytes

Bovine articular chondrocytes cultured in agarose gel in the presence of serum elaborated a highly organized extracellular matrix rich in proteoglycans and collagens. The cultures were evaluated quantitatively by radiosulfate labeling of proteoglycans, and by densitometry following staining with alcian blue. In addition, immunohistochemical methods were used to demonstrate the presence of several components of cartilage proteoglycan molecules. Treatment with Interleukin-1 (Il-1) or retinol resulted in diminished synthesis and enhanced catabolism of matrix proteoglycans, but the chondrocytes were more sensitive to human recombinant Il-1α than to Il-1β. Treatment with Il-1α or retinol resulted in a profound disorganization of the residual matrix around the majority of the chondrocytes, while Il-1β caused much less severe changes. Some variation in cellular response to Il-1α may result from the heterogeneity previously reported among articular chondrocytes.     

Influence of interleukin-1 on the morphology and proteoglycan metabolism of cultured bovine articular chondrocytes.

Bovine articular chondrocytes cultured in agarose gel in the presence of serum elaborated a highly organized extracellular matrix rich in proteoglycans and collagens. The cultures were evaluated quantitatively by radiosulfate labeling of proteoglycans, and by densitometry following staining with alcian blue. In addition, immunohistochemical methods were used to demonstrate the presence of several components of cartilage proteoglycan molecules. Treatment with Interleukin-1 (Il-1) or retinol resulted in diminished synthesis and enhanced catabolism of matrix proteoglycans, but the chondrocytes were more sensitive to human recombinant Il-1 alpha than to Il-1 beta. Treatment with Il-1 alpha or retinol resulted in a profound disorganization of the residual matrix around the majority of the chondrocytes, while Il-1 beta caused much less severe changes. Some variation in cellular response to Il-1 alpha may result from the heterogeneity previously reported among articular chondrocytes.     

Extracellular matrix and cell surface as determinants of connective tissue differentiation

This paper reviews in vitro studies, largely from the author's laboratory, concerning the conditions that are permissive for the differentiation of limb bud mesenchymal cells into chondrocytes. In high-density cell culture, even in a defined medium, the same normal sequence of events that is found in vivo in developing cartilage is also observed. This system can be used to study heritable disorders in model systems such as in mutant mouse embryos. In addition, single mesenchymal cells can differentiate into hypertrophic chondrocytes in hydrated collagen gel or agarose cultures. A rounded cell shape promotes chondrogenesis, while a flattened cell shape promotes fibroblast differentiation. The actin cytoskeleton is shown to play a central role in regulating connective tissue cell differentiation. By use of such cell culture manipulations, it is now possible to grow large numbers of fibroblastic cells from human biopsy material for storage and to carry out experimental studies after re-expression of chondrogenesis in gel cultures. It is suggested that cytoskeletal-extracellular matrix interactions play a fundamental role in connective tissue differentiation. Matrix receptors might be developmentally regulated and modify epithelial effects on mesenchymal cells. In this way mesenchymal cells differentiate in a highly organized manner in spatial and temporal terms.     

Prolonged exposure of human chondrocytes to ascorbic acid modifies cellular behavior in an agarose gel

Using an agarose gel culture system, the response of adult human chondrocytes to prolonged exposure of ascorbic acid was evaluated using histochemical, immunocytochemical and morphological techniques. The response of these cells to ascorbic acid was different from those previously reported in the literature. Many chondrocytes branched within the agarose gel with continued exposure to ascorbic acid while other chondrocytes maintained a round configuration typical of chondrocytes in vivo. Fibronectin and type I collagen were closely associated with the cell processes of the branching cells. Type II collagen and an alcian blue-staining matrix were associated with the rounded cells but not with the branched cells. These data suggest that the chondrocytes are able to express both dedifferentiated and redifferentiated phenotypes with ascorbic acid under these culture conditions. In addition, human chondrocytes were cultured in a collagen gel and began branching within 1 hour of culture. It is possible that an accumulation of type I collagen in the pericellular matrix of ascorbic acid treated cultures may enhance and explain the branching seen in these cultures. Studies by others have indicated that ascorbic acid may enhance, reduce, and/or modify the cartilage matrices produced by chondrocytes. These controversial reports in the literature are presumably due to variations between species and the culture methods employed. © 1994 Wiley-Liss, Inc.     

In-vitro culture of human chondrocytes from adult subjects

An agarose gel matrix was utilized to grow chondrocytes from human donors of various ages in cell culture. The chondrocytes produced the pericellular matrix characteristic for such cells and synthesized collagen type II as well as glyco-saminoglycans. The latter exhibit the typical distribution pattern of the respective articular cartilage matrix. The electron-microscopic appearance of the cultured chondrocytes closely resembles that of chondrocytes in sections of the original cartilage.     

Immunohistochemical distribution patterns of collagen type II,chondroitin 4-sulfate,laminin and fibronectin in human nasal septal cartilage

Collagen type II, chondroitin 4-sulfate, laminin and fibronectin are major components of cartilage matrix. It is important to know their distribution patterns to evaluate relationships between cartilage cells and surrounding cartilage matrix. In the present study, we investigated localization patterns of these macromolecules in human nasal septal cartilage by immunohistochemical methods. Samples of human nasal septal cartilage were obtained from patients with nasal septum deviations who underwent septoplastic operation and were prepared for immunohistochemical examination. Distribution patterns of cartilage matrix macromolecules correlated with those found in other cartilage tissues. Diffuse staining of collagen type II was found in the cartilage matrix, chondroitin 4-sulfate immunostaining was present in the cytoplasm and like a pericellular ring around chondrocytes. Laminin immunostaining was found in the cytoplasm of chondrocytes, and fibronectin was localized in the pericellular matrix and in capsules of human nasal septal cartilage. Moreover, fibronectin was also detected at high levels in the interconnecting segments between adjacent chondrons. In conclusion, similar localisation patterns of the components investigated in human septal cartilage as in other tissues indicate that these macromolecules may play a role in both cell-matrix adhesion and matrix-matrix cohesion in the pericellular microenvironment surrounding nasal septal cartilage chondrocytes as in other cartilage tissues.     

Type IIA procollagen: expression in developing chicken limb cartilage and human osteoarthritic articular cartilage.

Type IIA procollagen is an alternatively spliced product of the type II collagen gene and uniquely contains the cysteine (cys)-rich globular domain in its amino (N)-propeptide. To understand the function of type IIA procollagen in cartilage development under normal and pathologic conditions, the detailed expression pattern of type IIA procollagen was determined in progressive stages of development in embryonic chicken limb cartilages (days 5-19) and in human adult articular cartilage. Utilizing the antibodies specific for the cys-rich domain of the type IIA procollagen N-propeptide, we localized type IIA procollagen in the pericellular and interterritorial matrix of condensing pre-chondrogenic mesenchyme (day 5) and early cartilage (days 7-9). The intensity of immunostaining was gradually lost with cartilage development, and staining became restricted to the inner layer of perichondrium and the articular cap (day 12). Later in development, type IIA procollagen was re-expressed at the onset of cartilage hypertrophy (day 19). Different from type X collagen, which is expressed throughout hypertrophic cartilage, type IIA procollagen expression was transient and restricted to the zone of early hypertrophy. Immunoelectron microscopic and immunoblot analyses showed that a significant amount of the type IIA procollagen N-propeptide, but not the carboxyl (C)-propeptide, was retained in matrix collagen fibrils of embryonic limb cartilage. This suggests that the type IIA procollagen N-propeptide plays previously unrecognized roles in fibrillogenesis and chondrogenesis. We did not detect type IIA procollagen in healthy human adult articular cartilage. Expression of type IIA procollagen, together with that of type X collagen, was activated by articular chondrocytes in the upper zone of moderately and severely affected human osteoarthritic cartilage, suggesting that articular chondrocytes, which normally maintain a stable phenotype, undergo hypertrophic changes in osteoarthritic cartilage. Based on our data, we propose that type IIA procollagen plays a significant role in chondrocyte differentiation and hypertrophy during normal cartilage development as well as in the pathogenesis of osteoarthritis.     

Stirred flow bioreactor modulates chondrocyte growth and extracellular matrix biosynthesis in chitosan scaffolds

The aim of this study is to show the favorable effect of simple dynamic culture conditions on chondrogenesis of previously expanded human chondrocytes seeded in a macroporous scaffold with week cell-pore walls adhesion. We obtained enhanced chondrogenesis by the combination of chitosan porous supports with a double micro- and macro-pore structure and cell culture in a stirring bioreactor. Cell-scaffold constructs were cultured under static or mechanically stimulated conditions using an intermittent stirred flow bioreactor during 28 days. In static culture, the chondrocytes were homogeneously distributed throughout the scaffold pores; cells adhered to the scaffold pore walls, showed extended morphology and were able to proliferate. Immunofluorescense and biochemical assays showed abundant type I collagen deposition at day 28. However, the behavior of chondrocytes submitted to mechanical stimuli in the bioreactor was completely different. Mechanical loading influenced cell morphology and extracellular matrix composition. Under dynamic conditions, chondrocytes kept their characteristic phenotype and tended to form cell aggregates surrounded by a layer of the main components of the hyaline cartilage extracellular matrix, type II collagen, and aggrecan. An enhanced aggrecan and collagen type II production was observed in engineered cartilage constructs cultured under stirred flow compared with those cultured under static conditions.     

Smad signaling determines chondrogenic differentiation of bone-marrow-derived mesenchymal stem cells: inhibition of Smad1/5/8P prevents terminal differentiation and calcification

The aim of this study was to investigate the roles of Smad2/3 and Smad1/5/8 phosphorylation in transforming growth factor-beta-induced chondrogenic differentiation of bone-marrow-derived mesenchymal stem cells (BMSCs) to assess whether specific targeting of different Smad signaling pathways offers possibilities to prevent terminal differentiation and mineralization of chondrogenically differentiated BMSCs. Terminally differentiated chondrocytes produced in vitro by chondrogenic differentiation of BMSCs or studied ex vivo during murine embryonic limb formation stained positive for both Smad2/3P and Smad1/5/8P. Hyaline-like cartilage produced in vitro by articular chondrocytes or studied in ex vivo articular cartilage samples that lacked expression for matrix metalloproteinase 13 and collagen X only expressed Smad2/3P. When either Smad2/3 or Smad1/5/8 phosphorylation was blocked in BMSC culture by addition of SB-505124 or dorsomorphin throughout culture, no collagen II expression was observed, indicating that both pathways are involved in early chondrogenesis. Distinct functions for these pathways were demonstrated when Smad signaling was blocked after the onset of chondrogenesis. Blocking Smad2/3P after the onset of chondrogenesis resulted in a halt in collagen II production. On the other hand, blocking Smad1/5/8P during this time period resulted in decreased expression of matrix metalloproteinase 13, collagen X, and alkaline phosphatase while allowing collagen II production. Moreover, blocking Smad1/5/8P prevented mineralization. This indicates that while Smad2/3P is important for continuation of collagen II deposition, Smad1/5/8 phosphorylation is associated with terminal differentiation and mineralization.     

Clonal populations of chondrocytes with progenitor properties identified within human articular cartilage

The aim of the present study was to identify and characterize progenitor properties of human articular chondrocytes selected by using agarose suspension culture. In this chondrogenic selective culture condition, about 3.6% of seeded surplus chondrocytes from patients undergoing articular chondrocyte transplantation proliferated and formed cell clusters after 6 weeks. Phase-contrast microscopy and transmission electron microscopy revealed four different types of cell clusters differing in cellular content and matrix production. Based on their morphological features, they were named the homogenous (H), the homogenous matrix (HM), the differentiated matrix (DM) and the differentiated (D) cell clusters. All cell clusters showed positive safranin O staining, and matrix was positive for antibodies detecting type II collagen and aggrecan. The clusters were further demonstrated to express the genes for fibroblast growth factor receptor 3, type IIA collagen and type IIB collagen, while type X collagen was not expressed. After subcloning, the H and HM clusters demonstrated the best proliferative capacity. Chondrocytes from these two cell clusters also showed phenotypic plasticity in chondrogenic, adipogenic as well as osteogenic assays. This study demonstrates that existing subpopulations of cells with chondroprogenitor properties can be isolated from human adult articular cartilage using agarose suspension cultures.     

The Phenotypic Characterization of A13/BACii, a Novel Bovine Chondrocytic Cell Line with Differentiation Potential

In cartilage research bovine articular cartilage is used as an alternative to human tissue. However, animal material is subject to availability and primary cultures undergo senescence, limiting their use. Here we report the immortalization of primary bovine chondrocytes, which could be used as a surrogate for freshly isolated chondrocytes. Chondrocytes were isolated from cartilage explants and immortalized using 1.0 μg/ml benzo[alpha]pyrene. For 3-dimensional culture, chondrocytes were resuspended in 0.5% low-melt agarose at high density (HD) and cultured for 24 h prior to determining changes in expression profile and morphology. A13/BACii chondrocytes acquired a 'flat' irregular morphology and a foetal-like cell volume (1,509.59 ± 182.04 μm(3)). The human cell line C-20/A4 showed a statistically similar volume and length to A13/BACii. Two-dimensional-cultured A13/BACii expressed elevated levels of type I collagen (col1), reduced levels of type II collagen (col2) compared to freshly isolated chondrocytes and an overall col2 to col1 expression ratio (col2:col1) of 0.11 ± 0.01. Upon 3-dimensional encapsulation, there was a significant rise in col2 expression in both A13/BACii and C-20/A4, suggesting a capacity for redifferentiation in both cell lines with a return of col2:col1 values of A13/BACii to values previously observed in primary chondrocytes. A13/BACii chondrocytes expressed aggrecan, matrix metalloproteinase (MMP)-3, MMP-9 and MMP-13, further supporting indications of the differentiated phenotype. Here we report the creation of a novel chondrocytic cell line and demonstrate its strong potential for redifferentiation upon HD 3-dimensional encapsulation, providing an alternative to conventional dedifferentiated cell lines and primary culture.     

Low oxygen tension enhances chondroinduction by demineralized bone matrix in human dermal fibroblasts in vitro

Endochondral bone formation is induced by demineralized bone powder (DBP) when DBP is implanted subcutaneously in rodents. Previously, we developed an in vitro model of this process, wherein human dermal fibroblasts (hDFs) differentiate to chondrocytes when cultured in a three-dimensional porous collagen sponge containing DBP. In other studies, medium perfusion was beneficial in maintaining phenotype and viability of many cell types in plain porous collagen sponges, including fibroblasts, bone marrow stromal cells, osteoblasts, and epidermal cells. In contrast, medium perfusion inhibited chondrogenesis by articular chondrocytes; reduction of oxygen tension to 5%, however, restored chondrogenesis. These observations are consistent with the fact that in vivo cartilage is avascular and relatively hypoxic compared with other vascularized tissues. In this study, we tested the hypothesis that low oxygen tension (hypoxia, 5% oxygen) would enhance induced chondrogenesis in hDFs cultured with DBP. As expected, hypoxia upregulated hypoxia-inducible factor-1alpha in hDFs in all conditions (i.e. +/- perfusion, +/- DBP). Hypoxia increased accumulation of cartilage-specific matrix chondroitin 4-sulfate in hDFs, but only in the presence of DBP (165%, compared to normoxia, p < 0.05). Hypoxia did not appear to have detrimental effects on cell viability and proliferation. In sum, hypoxia enhanced cartilage matrix accumulation by hDFs cultured with DBP. These defined conditions can optimize the use of dermal fibroblasts for cartilage tissue engineering.     

Genetic manipulation of human mesenchymal progenitors to promote chondrogenesis using "bead-in-bead" polysaccharide capsules

Articular cartilage defects arising from trauma or degenerative diseases fail to repair spontaneously. We have adopted a non-viral gene delivery and tissue engineering strategy, in which Sox-9 transfected human mesenchymal progenitors have been encapsulated within alginate/chitosan polysaccharide capsules to promote chondrogenesis. Human bone marrow stromal cells and articular chondrocytes were transfected with flag-tagged Sox-9 plasmid and after 7 days in static culture, large regions of cell-generated matrix containing cartilage proteoglycans were observed as confirmed by positive Alcian blue staining and Sox-9 immunohistochemistry. Further, after 28 days, in vitro and in vivo, samples encapsulated with Sox-9 transfected cells demonstrated large regions of cartilaginous matrix as confirmed by positive Alcian blue staining, Sox-9 and type-II collagen immunohistochemistry, absent in samples encapsulated with untransfected cells. Extracted protein from in vivo constructs was further assessed by western blot analysis and positive expression of Sox-9 and type-II collagen was observed in Sox-9 transfected constructs which was absent in untransfected cells. Regions of cartilage-like matrix were significantly increased in Sox-9 constructs in comparison with untransfected constructs, confirming Sox-9 gene delivery enhances chondrogenesis in targeted cell populations, outlining the potential to promote cartilaginous construct formation with therapeutic implications for regeneration of human articular cartilage tissue defects.     

Structural colocalisation of type VI collagen and fibronectin in agarose cultured chondrocytes and isolated chondrons extracted from adult canine tibial cartilage

Cell–matrix and matrix–matrix interactions are of critical importance in regulating the development, maintenance and repair of articular cartilage. In this study, we examined the structural colocalisation of type VI collagen and fibronectin in isolated chondrons and long-term agarose cultured chondrocytes extracted from normal adult canine articular cartilage. Using double labelling immunohistochemistry in conjunction with dual channel confocal microscopy and digital image processing we demonstrate that type VI collagen and fibronectin are distributed in a similar staining pattern and are colocalised at the surface of cultured chondrocytes and isolated chondrons. The results suggest that type VI collagen and fibronectin may play a role in both cell–matrix adhesion and matrix–matrix cohesion in the pericellular microenvironment surrounding articular cartilage chondrocytes.     

Feedback control of selected biosynthetic activities of chondrocytes in culture

The syntheses of proteoglycans and proteins by chondrocytes from the Swarm rat chondrosarcoma in primary cultures were modulated on the addition of matrical molecules. In the presence of hyaluronan, collagen or proteoglycan aggregates the synthesis of proteoglycans was depressed. The synthesis of collagen was also depressed in the presence of hyaluronan or collagen. In the presence of proteoglycan monomers, the incorporation of 35S-sulfate was enhanced in proportion to the concentration of the additive in the medium; the synthesis of protein was unaffected. The proteoglycan monomers synthesized in the presence of proteoglycan monomers were larger than those synthesized in their absence. In combinations, the exogenous macromolecules did not affect the selected biosynthetic activities to an extent greater than that which they exerted separately. The data suggest, however, that the proteoglycan monomers can counteract the inhibitory effects of the macromolecules which are inhibitory. The data, moreover, suggest that the chondrocytes of the Swarm rat chondrosarcoma have the potential to discriminate between proteoglycan monomers produced by self and those produced by chondrocytes of hyaline cartilages.     

Analysis of N-cadherin function in limb mesenchymal chondrogenesis in vitro.

During embryonic limb development, cartilage formation is presaged by a crucial mesenchymal cell condensation phase. N-Cadherin, a Ca2+ -dependent cell-cell adhesion molecule, is expressed in embryonic chick limb buds in a spatiotemporal pattern suggestive of its involvement during cellular condensation; functional blocking of N-cadherin homotypic binding, by using a neutralizing monoclonal antibody, results in perturbed chondrogenesis in vitro and in vivo. In high-density micromass cultures of embryonic limb mesenchymal cells, N-cadherin expression level is high during days 1 and 2, coincident with active cellular condensation, and decreases upon overt chondrogenic differentiation from day 3 on. In this study, we have used a transfection approach to evaluate the effects of gain- and loss-of-function expression of N-cadherin constructs on mesenchymal condensation and chondrogenesis in vitro. Chick limb mesenchymal cells were transfected by electroporation with recombinant expression plasmids encoding wild-type or two mutant extracellular/cytoplasmic deletion forms of N-cadherin. Expression of the transfected N-cadherin forms showed a transient profile, being high on days 1-2 of culture, and decreasing by day 3, fortuitously coincident with the temporal profile of endogenous N-cadherin gene expression. Examined by means of peanut agglutinin (PNA) staining for condensing precartilage mesenchymal cells, cultures overexpressing wild-type N-cadherin showed enhanced cellular condensation on culture days 2 and 3, whereas expression of the deletion mutant forms (extracellular/cytoplasmic) of N-cadherin resulted in a decrease in PNA staining, suggesting that a complete N-cadherin protein is required for normal cellular condensation to occur. Subsequent chondrogenesis was also affected. Cultures overexpressing the wild-type N-cadherin protein showed enhanced chondrogenesis, indicated by increased production of cartilage matrix (sulfated proteoglycans, collagen type II, and cartilage proteoglycan link protein), as well as increased cartilage nodule number and size of individual nodules, compared with control cultures and cultures transfected with either of the two mutant N-cadherin constructs. These results demonstrate that complete N-cadherin function, at the levels of both extracellular homotypic binding and cytoplasmic linkage to the cytoskeleton by means of the catenin complex, is required for chondrogenesis by mediating functional mesenchymal cell condensation.     

Differentiation capacity of human chondrocytes embedded in alginate matrix

Healing capacity of cartilage is low. Thus, cartilage defects do not regenerate as hyaline but mostly as fibrous cartilage which is a major drawback since this tissue is not well adapted to the mechanical loading within the joint. During in vitro cultivation in monolayers, chondrocytes proliferate and de-differentiate to fibroblasts. In three-dimensional cell cultures, de-differentiated chondrocytes could re-differentiate toward the chondrogenic lineage and re-express the chondrogenic phenotype. The objective of this study was to characterize the mesenchymal stem cell (MSC) potential of human chondrocytes isolated from articular cartilage. Furthermore, the differentiation capacity of human chondrocytes in three-dimensional cell cultures was analyzed to target differentiation direction into hyaline cartilage. After isolation and cultivation of chondrogenic cells, the expression of the MSC-associated markers: cluster of differentiation (CD)166, CD44, CD105, and CD29 was performed by flow cytometry. The differentiation capacity of human chondrocytes was analyzed in alginate matrix cultured in Dulbecco?s modified eagle medium with (chondrogenic stimulation) and without (control) chondrogenic growth factors. Additionally, the expression of collagen type II, aggrecan, and glycosaminoglycans was determined. Cultivated chondrocytes showed an enhanced expression of the MSC-associated markers with increasing passages. After chondrogenic stimulation in alginate matrix, the chondrocytes revealed a significant increase of cell number compared with unstimulated cells. Further, a higher synthesis rate of glycosaminoglycans and a positive collagen type II and aggrecan immunostaining was detected in stimulated alginate beads. Human chondrocytes showed plasticity whilst cells were encapsulated in alginate and stimulated by growth factors. Stimulated cells demonstrated characteristics of chondrogenic re-differentiation due to collagen type II and aggrecan synthesis.