Effects of exogenous IGF-1 delivery on the early expression of IGF-1 signaling molecules by alginate embedded chondrocytes

Cartilage tissue engineering remains a significant challenge for both researchers and clinicians. Many strategic approaches, such as the delivery of growth factors to an in vitro cultured cartilage construct, continue to receive significant attention. However, the effects of delivering exogenous signaling molecules on endogenous signaling pathways within an engineered tissue are not well understood. In order to address this concern, we have investigated how the delivery of insulin-like growth factor-1 (IGF-1, delivered at concentrations of 0, 10, 50, and 100 ng/mL) affects the endogenous expression of IGF-1, its receptor (IGF-1R), and a well known IGF-1 binding protein (IGFBP-3) by articular chondrocytes embedded in alginate hydrogels over 8 days. To the best of our knowledge, this is the first report of delivery effects upon endogenous signal expression in a three-dimensional system relevant to tissue engineering objectives. Results showed significant differences in mRNA expression of IGF-1, IGF-1R, type II collagen, and type I collagen by day 8 between the induced versus noninduced IGF-1 groups. At day 8, the induced IGF-1 groups expressed IGF-1 mRNA four times lower than the 0 ng/mL IGF-1 group. Further, the IGF-1R mRNA expression was five times higher for the groups exposed to exogenous IGF-1 versus the 0 ng/mL IGF-1 case. Interestingly, the expression of IGFBP-3 decreased for all groups. Type II collagen expression was the highest and type I collagen was the lowest for the IGF-1 delivered samples. Finally, the different concentrations of IGF-1 investigated did not demonstrate significantly different trends in mRNA expression levels. Overall, results indicate that exogenous IGF-1 delivery does affect signaling molecule expression by chondrocytes embedded in alginate hydrogels, particularly downregulating the delivered signal while upregulating its receptor.     

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.     

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.     

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.     

Mutant WISP3 triggers the phenotype shift of articular chondrocytes by promoting sensitivity to IGF-1 hypothesis of spondyloepiphyseal dysplasia tarda with progressive arthropathy (SEDT-PA)

This article introduces the hypothesis that mutant WISP3 (Wnt1 inducible secreted protein-3) triggers the phenotype shift of the chondrocytes, especially in the articular chondrocytes, by promoting sensitivity to IGF-1 (insulin-like growth factor 1), and results in chondrocytes apoptosis in SEDT-PA. SEDT-PA is also referred to as progressive pseudorheumatoid dysplasia (PPD), arthropathy progressive pseudorheumatoid of childhood (APPRC). Evidence for the hypothesis is based on the following indications: (1) SEDT-PA is caused by mutations of the WISP3 gene. WISP3 encodes a domain that bears homology to the amino-terminal domain of the insulin-like growth factor binding proteins (IGFBPs). (2) IGF-1 enhances chondrocyte hypertrophy by insulin-like actions. WISP3 can up-regulate the expression of type II collagen. When chondrocytes become hypertrophic, they reduce the expression of types II and IX collagen. (3) The chondrocytes in the normal articular cartilage maintain a stable phenotype. These cells exhibit no mitotic activity, low matrix synthesis and low degradation. But articular chondrocytes could react to certain stimuli such as IGF-1. (4) The loss of WISP3 expression alters the phenotype of the breast epithelium and promotes motility and invasion. The WISP3-deficient cells are extremely sensitive to the growth stimulatory effects of IGF-1. (5) The action of IGF-I is inhibited by IGFBPs, both in articular chondrocytes and in the normal breast epithelium. In conclusion, the mutant WISP3 lose is the function of inhibiting IGF-1 and disturbs the maintenance of a stable phenotype in articular chondrocytes. So, the articular chondrocytes undergo hypertrophic and terminal differentiation apoptosis. The precise mechanism of WISP3 function during postnatal cartilage growth and homeostasis is not clear yet. This hypothesis provides a new clue on the present mechanism study on SEDT-PA. If verified, this new concept may lead to a novel pathogenesis of SEDT-PA.     

The potential of IGF-1 and TGFbeta1 for promoting "adult" articular cartilage repair: an in vitro study

Research into articular cartilage repair, a tissue unable to spontaneously regenerate once injured, has focused on the generation of a biomechanically functional repair tissue with the characteristics of hyaline cartilage. This study was undertaken to provide insight into how to improve ex vivo chondrocyte amplification, without cellular dedifferentiation for cell-based methods of cartilage repair. We investigated the effects of insulin-like growth factor 1 (IGF-1) and transforming growth factor beta 1 (TGFbeta1) on cell proliferation and the de novo synthesis of sulfated glycosaminoglycans and collagen in chondrocytes isolated from skeletally mature bovine articular cartilage, whilst maintaining their chondrocytic phenotype. Here we demonstrate that mature differentiated chondrocytes respond to growth factor stimulation to promote de novo synthesis of matrix macromolecules. Additionally, chondrocytes stimulated with IGF-1 or TGFbeta1 induced receptor expression. We conclude that IGF-1 and TGFbeta1 in addition to autoregulatory effects have differential effects on each other when used in combination. This may be mediated by regulation of receptor expression or endogenous factors; these findings offer further options for improving strategies for repair of cartilage defects.     

Comparison of phenotypic characterization between "alginate bead" and "pellet" culture systems as chondrogenic differentiation models for human mesenchymal stem cells

Chondrogenesis involves the recruitment of mesenchymal cells to differentiate into chondroblasts, and also the cells must synthesize a cartilage-specific extracellular matrix. There were two representative culture systems that promoted the chondrogenic differentiation of human mesenchymal stem cells. These systems were adaptations of the "pellet" culture system, which was originally described as a method for preventing the phenotypic modulation of chondrocytes, and the "alginate bead" culture system, which was used to maintain encapsulated cells at their differentiated phenotype over time, and also it was used to maintain the cells' proteoglycan synthesis at a rate similar to that of primary chondrocytes. We performed test on the differences of phenotypic characterization with the two methods of differentiating human mesenchymal stem cells into chondrocytes. The typical gene for articular cartilage, collagen type II, was more strongly expressed in the "alginate bead" system than in the "pellet" culture system, in addition, specific gene for hypertrophic cartilage, collagen type X, was more rapidly expressed in the "pellet" system than in "alginate bead" culture system. Therefore, the "alginate bead" culture system is a more phenotypical, practical and appropriate system to differentiate human mesenchymal stem cells into articular chondrocytes than the "pellet" culture system.     

Comparative phenotypic analysis of articular chondrocytes cultured within type I or type II collagen scaffolds

Among the existing repair strategies for cartilage injury, tissue engineering approach using biomaterials and chondrocytes offers hope for treatments. In this context, collagen-based biomaterials are good candidates as scaffolds for chondrocytes in cell transplantation procedures. These scaffolds are provided under different forms (gel or crosslinked sponge) made with either type I collagen or type I or type II atelocollagen molecules. The present study was undertaken to investigate how bovine articular chondrocytes sense and respond to differences in the structure and organization of these collagen scaffolds, over a 12-day culture period. When chondrocytes were seeded in the collagen scaffolds maintained in free-floating conditions, cells contracted gels to 40-60% and sponges to 15% of their original diameter. Real-time polymerase chain reaction analysis indicated that the chondrocyte phenotype, assessed notably by the ratio of COL2A1/COL1A2 mRNA and alpha10/alpha11 integrin subunit mRNA, was comparatively better sustained in type I collagen sponges when seeded at high cell density, also in type I atelocollagen gels. Besides, proteoglycan accumulation in the different scaffolds, as assessed by measuring the sulfated glycosaminoglycan content, was found be highest in type I collagen sponges seeded at high cell density. In addition, gene expression of matrix metalloproteinase-13 increased dramatically (up to 90-fold) in chondrocytes cultured in the different gels, whereas it remained stable in the sponges. Our data taken together reveal that type I collagen sponges seeded at high cell density represent a suitable material for tissue engineering of cartilage.     

Biosynthetic response of passaged chondrocytes in a type II collagen scaffold to mechanical compression

To investigate the potential utility of mechanical loading in articular cartilage tissue engineering, porous type II collagen scaffolds seeded with adult canine passaged chondrocytes were subjected to static and dynamic compressions of varying magnitudes (0-50% static strain) and durations (1-24 h), and at different times during culture (2-30 days postseeding). The effects of mechanical compression on the biosynthetic activity of the chondrocytes were evaluated by measuring the amount of (3)H-proline-labeled proteins and (35)S-sulfate-labeled proteoglycans that accumulated in the cell-scaffold construct and was released to the medium during the loading period. Similar to published results on loading of articular cartilage explants, static compression decreased protein and proteoglycan biosynthesis in a time- and dose-dependent manner (each p < 0.005), and selected dynamic compression protocols were able to increase rates of biosynthesis (p < 0.05). The main difference between the results seen for this tissue engineering system and cartilage explants was in the amount of newly synthesized matrix molecules that accumulated within the construct under dynamic loading, with less accumulating in the type II collagen scaffold. In summary, the general biosynthetic response of passaged chondrocytes in the porous type II collagen scaffolds is similar to that seen for chondrocytes in their native environment. Future work needs to be directed to modifications of the cell-seeded construct to allow for the capture of the newly synthesized matrix molecules by the scaffold.     

Retaining zonal chondrocyte phenotype by means of novel growth environments

The loss of phenotype in articular chondrocytes expanded in monolayer has been established as a possible contributor to the deficiencies associated with in vitro cartilage engineering and autologous cell transplantation procedures. We cultured zonal articular chondrocytes on tissue culture plastic, collagen II-coated polystyrene, and aggrecan-coated polystyrene in an effort to find a surface that can either prevent or slow the loss of phenotype. In addition, we encapsulated passaged cells in agarose to examine the effect of three-dimensional culture on redifferentiating zonal chondrocytes. We used real-time polymerase chain reaction to measure the relative gene expression levels of collagen I and II, aggrecan, and superficial zone protein over relevant passages (P0-P4). Results showed that tissue culture plastic and the collagen II-coated surface induced rapid loss of phenotype in zonal articular chondrocytes. The aggrecan-coated surface had a less detrimental effect on the chondrocytic phenotype of seeded cells, inducing gene expression characteristics comparable to those of agarose-encapsulated cells. Furthermore, when chondrocytes that had been previously passaged on a collagen II surface were placed on an aggrecan surface, the zonal cells showed a dramatic change in gene expression from fibroblastic to chondrocytic. These results indicate that a culture environment using aggrecan as a substratum or agarose as a scaffold is crucial to the development of phenotypically correct articular cartilage.     

Formation of cartilage matrix proteins by BMP-transfected murine mesenchymal stem cells encapsulated in a novel class of alginates

Proliferation and differentiation of wild-type, BMP-2 and BMP-4 transfected cells of C3H10T1/2, a mouse mesenchymal stem cell line that can differentiate into chondrocytes, were studied under monolayer (2D-) and encapsulation (3D-) conditions. Cells were encapsulated in a novel class of alginate. The alginate was of clinical grade (CG) because of complete removal of mitogenic and cytotoxic contaminants by chemical means. Compared to commercial alginates used so far for encapsulation it was characterized by ultra-high viscosity (UHV; viscosity of a 0.1% w/v solution of about 20 cP). In contrast to monolayer cultures, proliferation of cells was prevented when the cells were encapsulated in UHV/CG alginate at the same suspension density. As revealed by immunohistochemistry and quantitative RT-PCR, transfected and wild-type monolayer cells showed synthesis of type I collagen after transfer into differentiation medium, while culture in an alginate scaffold resulted in an upregulation of type II collagen and other hyaline cartilage proteins. BMP-4 transfected cells produced considerably more type II collagen than BMP-2 transfected and wild-type cells. BMP-4 transfected cells were also characterized by type I collagen production up to Day 10 and exhibited transient alkaline phosphatase activity levels that were much higher than the peak values observed for the other two cell lines. The coincidence of the ALP peak values with downregulation of type I collagen in BMP-4 transfected cells suggested that C3H10T1/2 cells differentiate into chondrocytes via a chondroprogenitor-like cell.     

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.     

Effect of oxygen tension and alginate encapsulation on restoration of the differentiated phenotype of passaged chondrocytes.

The implantation of laboratory-grown tissue offers a valuable alternative approach to the treatment of cartilage defects. Procuring sufficient cell numbers for such tissue-engineered cartilage is a major problem since amplification of chondrocytes in culture typically leads to loss of normal cell phenotype yielding cartilage of inferior quality. In an effort to overcome this problem, we endeavored to regain the differentiated phenotype of chondrocytes after extensive proliferation in monolayer culture by modulating cell morphology and oxygen tension towards the in vivo state. Passaged cells were encapsulated in alginate hydrogel in an effort to regain the more rounded shape characteristic of differentiated chondrocytes. These cultures were exposed to reduced (5%-i.e., physiological), or control (20%) oxygen tensions. Both alginate encapsulation and reduced oxygen tension significantly upregulated collagen II and aggrecan core protein expression (differentiation markers). In fact, after 4 weeks in alginate at 5% oxygen, differentiated gene expression was comparable to primary chondrocytes. Collagen I expression (dedifferentiation marker) decreased dramatically after alginate entrapment, while reduced oxygen tension had no effect. It is concluded that alginate encapsulation and reduced oxygen tension help restore key differentiated phenotypic markers of passaged chondrocytes. These findings have important implications for cartilage tissue engineering, since they enable the increase in differentiated cell numbers needed for the in vitro development of functional cartilaginous tissue suitable for implantation.     

Cartilage tissue engineering using human auricular chondrocytes embedded in different hydrogel materials

To seek a suitable scaffold for cartilage tissue engineering, we compared various hydrogel materials originating from animals, plants, or synthetic peptides. Human auricular chondrocytes were embedded in atelopeptide collagen, alginate, or PuraMatrix, all of which are or will soon be clinically available. The chondrocytes in the atelopeptide collagen proliferated well, while the others showed no proliferation. A high-cell density culture within each hydrogel enhanced the expression of collagen type II mRNA, when compared with that without hydrogel. By stimulation with insulin and BMP-2, collagen type II and glycosaminoglycan were significantly accumulated within all hydrogels. Chondrocytes in the atelopeptide collagen showed high expression of beta1 integrin, seemingly promoting cell-matrix signaling. The N-cadherin expression was inhibited in the alginate, implying that decrease in cell-to-cell contacts may maintain chondrocyte activity. The matrix synthesis in PuraMatrix was less than that in others, while its Young's modulus was the lowest, suggesting a weakness in gelling ability and storage of cells and matrices. Considering biological effects and clinical availability, atelopeptide collagen may be accessible for clinical use. However, because synthetic peptides can control the risk of disease transmission and immunoreactivities, some improvement in gelling ability would provide a more useful hydrogel for ideal cartilage regeneration.     

In vitro cartilage tissue formation by Co-culture of primary and passaged chondrocytes

Passaging chondrocytes to increase cell number is one way to overcome the major limitation to cartilage tissue engineering, which is obtaining sufficient numbers of chondrocytes to form large amounts of tissue. Because neighboring cells can influence cell phenotype and because passaging induces dedifferentiation, we examined whether coculture of primary and passaged bovine articular chondrocytes in 3-dimensional culture would form cartilage tissue in vitro. Chondrocytes passaged in monolayer culture up to 4 times were mixed with primary (nonpassaged) chondrocytes (5-40% of total cell number) and grown on filter inserts for up to 4 weeks. Passaged cells alone did not form cartilage, but with the addition of increasing numbers of primary chondrocytes, up to 20%, there was an increase in cartilage tissue formation as determined histologically and biochemically and demonstrated by increasing proteoglycan and collagen accumulation. The passaged cells appeared to be undergoing redifferentiation, as indicated by up-regulation of aggrecan, type II collagen, and SOX9 gene expression and decreased type I collagen expression. This switch in collagen type was confirmed using Western blots. Confocal microscopy showed that fluorescently labeled primary cells were distributed throughout the tissue. This coculture approach could provide a new way to solve the problem of limited cell number for cartilage tissue engineering.     

Comparison of different chondrocytes for use in tissue engineering of cartilage model structures

This study compares bovine chondrocytes harvested from four different animal locations--nasoseptal, articular, costal, and auricular--for tissue-engineered cartilage modeling. While the work serves as a preliminary investigation for fabricating a human ear model, the results are important to tissue- engineered cartilage in general. Chondrocytes were cultured and examined to determine relative cell proliferation rates, type II collagen and aggrecan gene expression, and extracellular matrix production. Respective chondrocytes were then seeded onto biodegradable poly(L-lactide-epsilon-caprolactone) disc-shaped scaffolds. Cell-copolymer constructs were cultured and subsequently implanted in the subcutaneous space of athymic mice for up to 20 weeks. Neocartilage development in harvested constructs was assessed by molecular and histological means. Cell culture followed over periods of up to 4 weeks showed chondrocyte proliferation from the tissue sources varied, as did levels of type II collagen and aggrecan gene expression. For both genes, highest expression was found for costal chondrocytes, followed by nasoseptal, articular, and auricular cells. Retrieval of 20-week discs from mice revealed changes in construct dimensions with different chondrocytes. Greatest disc diameter was found for scaffolds seeded with auricular chondrocytes, followed by those with costal, nasoseptal, and articular cells. Greatest disc thickness was measured for scaffolds containing costal chondrocytes, followed by those with nasoseptal, auricular, and articular cells. Retrieved copolymer alone was smallest in diameter and thickness. Only auricular scaffolds developed elastic fibers after 20 weeks of implantation. Type II collagen and aggrecan were detected with differing expression levels on quantitative RT-PCR of discs implanted for 20 weeks. These data demonstrate that bovine chondrocytes obtained from different cartilaginous sites in an animal may elicit distinct responses during their respective development of a tissue-engineered neocartilage. Thus, each chondrocyte type establishes or maintains its particular developmental characteristics, and this observation is critical in the design and elaboration of any tissue-engineered cartilage model.     

Transduction of passaged human articular chondrocytes with adenoviral, retroviral, and lentiviral vectors and the effects of enhanced expression of SOX9

Chondrocytes form and maintain the extracellular matrix of cartilage. The cells can be isolated from cartilage for applications such as tissue engineering, but their expansion in monolayer culture causes a progressive loss of chondrogenic phenotype. In this work, we have investigated the isolation of human articular chondrocytes from osteoarthritic (OA) cartilage at joint replacement, their expansion in monolayer culture, and their transduction with adenoviral, retroviral, and lentiviral vectors, using the gene encoding green fluorescent protein as a marker gene. The addition of growth factors (transforming growth factor beta(1), fibroblast growth factor 2, and platelet-derived growth factor BB) during cell culture was found to greatly increase cell proliferation and thereby to selectively enhance the efficiency of transduction with retrovirus. With adenoviral and lentiviral vectors the transduction efficiency achieved was 95 and 85%, respectively. Using growth factor-supplemented medium with a retroviral vector, efficiency in excess of 80% was achieved. The expression was stable for several months with both retrovirus and lentivirus when analyzed by fluorescence-activated cell-sorting flow analysis and immunoblotting. Transduction with SOX9 was investigated as a method to reinitiate cartilage matrix gene expression in passaged human OA chondrocytes. Endogenous collagen II expression (both mRNA and protein) was increased in monolayer culture using both adenoviral and retroviral vectors. Furthermore, collagen II gene expression in chondrocytes retrovirally transduced with SOX9 was stimulated by alginate bead culture, whereas in control chondrocytes it was not. These results demonstrated methods for rapid expansion and highly efficient transduction of human OA chondrocytes and the potential for the recovery of key features of chondrocyte phenotype by transduction with SOX9.     

Effect of chondrocyte passage number on histological aspects of tissue-engineered cartilage

Transplantation of cultured chondrocytes can regenerate cartilage tissue in cartilage defects. This method requires serial cell passages to expand chondrocytes to a large number of cells for transplantation. However, as chondrocytes are expanded in number in monolayer culture, the cells gradually lose their differentiated phenotype and may not form cartilage tissue. This study investigated whether chondrocytes cultured through various passages maintain their potential to reexpress a chondrogenic phenotype in three-dimensional scaffolds and form cartilage tissue in vitro and in vivo. The growth rate, viability, synthesis of collagen type I and II, and apoptotic activity of chondrocytes with passage number of 1, 2 and 5 were compared during in vitro culture. As the passage number increased, the cell growth rate and viability decreased and apoptotic cell increased. Passage 2 chondrocytes exhibited a high expression of collagen type II and a low expression of collagen type I. In contrast, passage 5 chondrocytes exhibited a low expression of collagen type II and a high expression of collagen type I, indicating chondrocyte dedifferentiation. To examine the ability of chondrocytes to regenerate cartilage tissues in vitro and in vivo, chondrocytes were expanded in vitro to passage number of 1 or 5, seeded onto biodegradable polymer scaffolds, and maintained in vitro or implanted into subcutaneous spaces of athymic mice for 1 month. Histological and immunohistochemical analyses of cartilage tissues engineered in vitro and in vivo with passage 1 chondrocytes showed mature and well-formed cartilage and the presence of highly sulfated glycosaminoglycans and type II collagen, a collagen type produced by differentiated chondrocytes. In contrast, tissues engineered in vitro and in vivo with passage 5 chondrocytes did not have chondrocyte morphology or cartilage-specific extracellular matrices (i.e., glycosaminoglycans and type II collagen). The results of this study show that chondrocyte passage number is an important factor affecting the quality of cartilage tissue-engineered with the chondrocytes, and that chondrocytes.