Dedifferentiation Of Articular Chondrocytes Research Articles

A novel modified culture medium for enhancing redifferentiation of chondrocytes for cartilage tissue engineering applications

The dedifferentiation of articular chondrocytes during in vitro expansion deteriorates the hyaline cartilage regeneration. Many approaches have been developed to enhance the redifferentiation of chondrocytes. In this study, a new and effective protocol to improve the redifferentiation of porcine chondrocytes in a pellet form was established. Pellets were initially treated in the modified culture media containing ternary mixtures, binary mixtures, or single reagents of sodium citrate (SCi), sodium chloride (SCh), and ethylenediaminetetraacetic acid (EDTA) at varied concentrations during the first 3 days of culture, followed by a normal culture medium until 21 days. Viability, proliferation, cartilaginous gene expression, extracellular matrix formation, and morphology of treated cell pellets were comparatively examined. Chondrocytes exposed to SCi, SCh, and EDTA individually or in combinations of two or three chemicals were non-cytotoxic when the concentration ranges of the chemicals were 1.83-2.75, 5.00-7.50, and 1.00-1.50 mM, respectively. Cells treated with the modified media containing EDTA alone and EDTA-containing mixtures enhanced glycosaminoglycan production as well as upregulated cartilaginous gene expression, despite their low proliferation rates. Overall, when all three reagents were in use, a pronounced synergistic effect on the activations of glycosaminoglycan accumulation and type II collagen production was explicitly observed at most, particularly when cells were cultured in the medium containing SCi, SCh, and EDTA at concentrations of 2.20, 6.00, and 1.20 mM, respectively. With a use of this protocol, the redifferentiation of articular chondrocytes for regeneration of hyaline cartilage for tissue engineering applications could be readily achieved.

Inhibition of CD44 intracellular domain production suppresses bovine articular chondrocyte de-differentiation induced by excessive mechanical stress loading

CD44 fragmentation is enhanced in chondrocytes of osteoarthritis (OA) patients. We hypothesized that mechanical stress-induced enhancement of CD44-intracellular domain (CD44-ICD) production plays an important role in the de-differentiation of chondrocytes and OA. This study aimed to assess the relationship between CD44-ICD and chondrocyte gene expression. Monolayer cultured primary bovine articular chondrocytes (BACs) were subjected to cyclic tensile strain (CTS) loading. ADAM10 inhibitor (GI254023X) and γ-secretase inhibitor (DAPT) were used to inhibit CD44 cleavage. In overexpression experiments, BACs were electroporated with a plasmid encoding CD44-ICD. CTS loading increased the expression of ADAM10 and subsequent CD44 cleavage, while decreasing the expression of SOX9, aggrecan, and type 2 collagen (COL2). Overexpression of CD44-ICD also resulted in decreased expression of these chondrocyte genes. Both GI254023X and DAPT reduced the production of CD44-ICD upon CTS loading, and significantly rescued the reduction of SOX9 expression by CTS loading. Chemical inhibition of CD44-ICD production also rescued aggrecan and COL2 expression following CTS loading. Our findings suggest that CD44-ICD is closely associated with the de-differentiation of chondrocytes. Excessive mechanical stress loading promoted the de-differentiation of BACs by enhancing CD44 cleavage and CD44-ICD production. Suppression of CD44 cleavage has potential as a novel treatment strategy for OA.

Ginsenoside Rg1‐modified PHBV fibrous scaffold reduces interleukin‐1 beta‐induced dedifferentiation of articular chondrocytes

ABSTRACTInflammatory environment in the end‐stage osteoarthritis (OA) cartilage defects greatly limits the effectiveness of engineered cartilage. It is critical that developing scaffolds intend to be applied in cartilage tissue engineering (CTE) under this pathological condition. In this study, poly(3‐hydroxybutyric acid‐137‐3‐hydroxyvaleric acid) (PHBV) fibrous scaffolds modified by ginsenoside Rg1 could be able to alleviate the dedifferentiation of articular chondrocytes caused by interleukin‐1 beta (IL‐1β), a key inflammatory factor in the pathogenesis of OA. Followed by 5‐day incubation with culture media containing IL‐1β, articular chondrocytes cultured with ginsenoside Rg1‐modified PHBV fibrous scaffolds (PHBV‐g‐Rg1) displayed typical chondrocytic morphology compared with those cultured with PHBV fibrous scaffolds. By employing real‐time quantitative polymerase chain reaction assay, it demonstrated that PHBV‐g‐Rg1 fibrous scaffold reduced IL‐1β‐caused dedifferentiation to articular chondrocytes via enhancing the transcription of chondrocytic phenotype‐related genes and reducing the transcription of matrix degradation‐related genes. It suggests that ginsenoside Rg1‐modified PHBV fibrous scaffold would be potentially applied in CTE to treat cartilage defects under IL‐1β threat. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2020, 137, 48378.

Translational control maintains cartilage homeostasis and regulates osteoarthritis progression

Purpose: The unique properties of articular cartilage are defined by quiescent, differentiated articular chondrocytes whose homeostasis is maintained through a strict balance between anabolic and catabolic processes. This balance is shifted toward catabolic activities in osteoarthritis (OA), a disease that involves all joint tissues and disrupts the structural integrity of articular cartilage. In OA, articular chondrocytes become more hypertrophic-ike, and abnormally increase not only extracellular matrix (ECM) production but also the expression of ECM-degrading enzymes. While chondrocyte hypertrophy is indispensable for endochondral bone formation, in OA it mediates detrimental changes that lead to cartilage destruction. Our previous work established that translational control of gene expression is critical for dedifferentiation of articular chondrocytes in OA. We showed that fine-tuned control of protein synthesis by translational repressor 4E-BP (eIF4E-Binding Protein) is required for proper chondrocyte homeostasis. By binding to the m7-GTP cap-binding protein eIF4E, 4E-BP inhibits cap-dependent mRNA translation by restricting the incorporation of eIF4E into a multi-subunit initiation factor called eIF4F, which is needed to recruit 40S ribosomes to mRNA. We hypothesized that proper translational control is crucial for maintaining healthy cartilage phenotype. We therefore investigated expression of which genes is controlled (completely or partially) at the translational level. Methods: To identify the pool of mRNAs that are translationally control in OA cartilage we first compared translation efficiencies of mRNAs by analyzing polysome profiles of rat articular chondrocytes (RAC) untreated and treated with Interleukin 1β (IL-1β). Expression of several translationally regulated mRNAs was then compared to their expression in human and rodent OA cartilage. Additionally, the eIF4F inhibitor was used in vivo to determine if restriction of eIF4F activity affects OA progression in ACLT rat model. Results: We identified 617 mRNAs that are translationally regulated by IL-1β. One of them is the orphan receptor Nr4a1, whose protein but not mRNA level is significantly increased in OA cartilage in vivo, highlighting the exceptional potential of our approach for discovering new possible targets in OA cartilage. Modulating Nr4a1 expression in IL-1β-treated articular chondrocytes effects expression of MMP13, the metalloproteinase that has a predominant role in OA. Intra-articular injection of 4E1RCat, an inhibitor of cap-dependent translation not only precludes Fn and Nr4a upregulation in a rodent OA model, but also significantly delays cartilage degeneration in the ACLT rat model. Conclusions: We identified 617 mRNAs that are translationally regulated by IL-1β. One of them is the orphan receptor Nr4a1, whose protein but not mRNA level is significantly increased in OA cartilage in vivo, highlighting the exceptional potential of our approach for discovering new possible targets in OA cartilage. Modulating Nr4a1 expression in IL-1β-treated articular chondrocytes effects expression of MMP13, the metalloproteinase that has a predominant role in OA. Intra-articular injection of 4E1RCat, an inhibitor of cap-dependent translation not only precludes Fn and Nr4a upregulation in a rodent OA model, but also significantly delays cartilage degeneration in the ACLT rat model.

Simvastatin prevents articular chondrocyte dedifferentiation induced by nitric oxide by inhibiting the expression of matrix metalloproteinases 1 and 13.

Dedifferentiation of chondrocytes is the main character of cartilage degradation. Therefore the understanding of chondrocytes dedifferentiation is essential for arthritis therapy. However, the molecular mechanism of cartilage destroy is mostly unknown. In this work we show that simvastatin (SVT) inhibits dedifferentiation by nitric oxide by blocking the expression of matrix metalloproteinases 1 and 13. These effects of SVT on dedifferentiation suggest that SVT may be used as a drug for the cure of arthritis.

Influence of Mechanical Unloading on Articular Chondrocyte Dedifferentiation

Influence of Mechanical Unloading on Articular Chondrocyte Dedifferentiation

Influence of Mechanical Unloading on Articular Chondrocyte Dedifferentiation.

Due to the limited self-repair capacity of articular cartilage, the surgical restoration of defective cartilage remains a major clinical challenge. The cell-based approach, which is known as autologous chondrocyte transplantation (ACT), has limited success, presumably because the chondrocytes acquire a fibroblast-like phenotype in monolayer culture. This unwanted dedifferentiation process is typically addressed by using three-dimensional scaffolds, pellet culture, and/or the application of exogenous factors. Alternative mechanical unloading approaches are suggested to be beneficial in preserving the chondrocyte phenotype. In this study, we examined if the random positioning machine (RPM) could be used to expand chondrocytes in vitro such that they maintain their phenotype. Bovine chondrocytes were exposed to (a) eight days in static monolayer culture; (b) two days in static monolayer culture, followed by six days of RPM exposure; and, (c) eight days of RPM exposure. Furthermore, the experiment was also conducted with the application of 20 mM gadolinium, which is a nonspecific ion-channel blocker. The results revealed that the chondrocyte phenotype is preserved when chondrocytes go into suspension and aggregate to cell clusters. Exposure to RPM rotation alone does not preserve the chondrocyte phenotype. Interestingly, the gene expression (mRNA) of the mechanosensitive ion channel TRPV4 decreased with progressing dedifferentiation. In contrast, the gene expression (mRNA) of the mechanosensitive ion channel TRPC1 was reduced around fivefold to 10-fold in all of the conditions. The application of gadolinium had only a minor influence on the results. This and previous studies suggest that the chondrocyte phenotype is preserved if cells maintain a round morphology and that the ion channel TRPV4 could play a key role in the dedifferentiation process.

PEP-1-SIRT2-induced matrix metalloproteinase-1 and -13 modulates type II collagen expression via ERK signaling in rabbit articular chondrocytes

Matrix metalloproteinases (MMPs) are critical for the degradation of the extracellular matrix (ECM), which includes cartilage-specific collagen types I, II and XI. We previously found that PEP-1-sirtuin (SIRT)2 could induce dedifferentiation of articular chondrocytes; however, the underlying mechanisms remains unclear. We addressed this in the present study by examining the association between PEP-1-SIRT2 and the expression of MMP-1 and MMP-13 and type II collagen in rabbit articular chondrocytes. We found that PEP-1-SIRT2 increased MMP-1 and -13 expression in a dose- and time-dependent manner, as determined by western blotting. A similar trend in MMP-1 and -13 levels was observed in cultures during expansion to four passages. Pharmacological inhibition of MMP-1 and -13 blocked the PEP-1–SIRT2-induced decrease in type II collagen level. Phosphorylation of extracellular regulated kinase (ERK) was increased by PEP-1–SIRT2; however, treatment with the mitogen-activated protein kinase inhibitor PD98059 suppressed PEP-1–SIRT2-induced MMP-1 and -13 expression and dedifferentiation while restoring type II collagen expression in passage 2 cells. These results suggest that PEP-1–SIRT2 promotes MMP-induced dedifferentiation via ERK signaling in articular chondrocytes.

Chondrocytes, Mesenchymal Stem Cells, and Their Combination in Articular Cartilage Regenerative Medicine.

Articular cartilage (AC) is a highly organized connective tissue lining, covering the ends of bones within articulating joints. Its highly ordered structure is essential for stable motion and provides a frictionless surface easing load transfer. AC is vulnerable to lesions and, because it is aneural and avascular, it has limited self-repair potential which often leads to osteoarthritis. To date, no fully successful treatment for osteoarthritis has been reported. Thus, the development of innovative therapeutic approaches is desperately needed. Autologous chondrocyte implantation, the only cell-based surgical intervention approved in the United States for treating cartilage defects, has limitations because of de-differentiation of articular chondrocytes (AChs) upon in vitro expansion. De-differentiation can be abated if initial populations of AChs are co-cultured with mesenchymal stem cells (MSCs), which not only undergo chondrogenesis themselves but also support chondrocyte vitality. In this review we summarize studies utilizing AChs, non-AChs, and MSCs and compare associated outcomes. Moreover, a comprehensive set of recent human studies using chondrocytes to direct MSC differentiation, MSCs to support chondrocyte re-differentiation and proliferation in co-culture environments, and exploratory animal intra- and inter-species studies are systematically reviewed and discussed in an innovative manner allowing side-by-side comparisons of protocols and outcomes. Finally, a comprehensive set of recommendations are made for future studies.

MicroRNA-140 TargetsRALAand Regulates Chondrogenic Differentiation of Human Mesenchymal Stem Cells by Translational Enhancement ofSOX9andACAN

Lesions of articular cartilage do not heal spontaneously. One treatment strategy would be to make cartilage in the laboratory by directed chondrogenic differentiation of mesenchymal stem cells (MSCs). To promote our understanding of the molecular control of chondrogenesis, we have compared the changes in microRNAs (miRNAs) during in vitro chondrogenesis of MSCs with those observed in uncultured and dedifferentiated articular chondrocytes (ACs). Several miRNAs showed a reciprocal relationship during the differentiation of MSCs and dedifferentiation of ACs. miR-140-5p and miR-140-3p changed the most during in vitro chondrogenesis, they were the miRNAs most highly expressed in tissue-engineered chondrocytes, and they were also among the miRNAs most highly expressed in uncultured ACs. There was a 57% overlap for the 100 most highly expressed miRNAs in differentiated MSCs and uncultured ACs, but for other miRNAs, the expression pattern was quite different. We transiently and stably inhibited and overexpressed miR-140-5p and miR-140-3p in differentiating MSCs and dedifferentiating ACs, respectively, to describe global effects and identify and validate new targets. Surprisingly, SOX9 and aggrecan proteins were found to be downregulated in anti-miR-140 transduced differentiating MSCs despite unchanged mRNA levels. This suggests that miR-140 stimulates in vitro chondrogenesis by the upregulation of these molecules at the protein level. RALA, a small GTPase, was identified as a miR-140 target and knockdown experiments showed that RALA regulated SOX9 at the protein level. These observations shed new light on the effect of miR-140 for chondrogenesis in vitro and in vivo.

The roles of canonical and non-canonical Wnt signaling in human de-differentiated articular chondrocytes

Osteoarthritis is the most prevalent form of arthritis in the world and it is becoming a major public health problem. Osteoarthritic chondrocytes undergo morphological and biochemical changes that lead to de-differentiation. The involvement of signaling pathways, such as the Wnt pathway, during cartilage pathology has been reported. Wnt signaling regulates critical biological processes. Wnt signals are transduced through at least three intracellular signaling pathways including the canonical Wnt/β-catenin pathway, the Wnt/Ca2 + pathway and the Wnt/planar cell polarity pathway. We investigated the involvement of the Wnt canonical and non-canonical pathways in human articular chondrocyte de-differentiation in vitro. Human articular chondrocytes were cultured through four passages with no treatment, or with sFRP3 treatment, an inhibitor of Wnt pathways, or with DKK1 treatment, an inhibitor of the canonical pathway. Chondrocyte-secreted markers and Wnt pathway components were analyzed using western blotting and qPCR. Inhibition of the Wnt pathway showed that the canonical Wnt signaling probably is responsible for inhibition of collagen II expression, activation of metalloproteinase 13 expression and regulation of Wnt7a and c-jun expression during chondrocyte de-differentiation in vitro. Our results also suggest that expressions of eNOS, Wnt5a and cyclinE1 are regulated by non-canonical Wnt signaling.

Wnt signaling is involved in human articular chondrocyte de-differentiation in vitro

Osteoarthritis is the most prevalent form of arthritis in the world. Certain signaling pathways, such as the wnt pathway, are involved in cartilage pathology. Osteoarthritic chondrocytes undergo morphological and biochemical changes that lead to chondrocyte de-differentiation. We investigated whether the Wnt pathway is involved in de-differentiation of human articular chondrocytes in vitro. Human articular chondrocytes were cultured for four passages in the presence or absence of IL-1 in monolayer or micromass culture. Changes in cell morphology were monitored by light microscopy. Protein and gene expression of chondrocyte markers and Wnt pathway components were determined by Western blotting and qPCR after culture. After culturing for four passages, chondrocytes exhibited a fibroblast-like morphology. Collagen type II and aggrecan protein and gene expression decreased, while collagen type I, matrix metalloproteinase 13, and nitric oxide synthase expressions increased. Wnt molecule expression profiles changed; Wnt5a protein expression, the Wnt target gene, c-jun, and in Wnt pathway regulator, sFRP4 increased. Treatment with IL-1 caused chondrocyte morphology to become more filament-like. This change in morphology was accompanied by extinction of col II expression and increased col I, MMP13 and eNOS expression. Changes in expression of the Wnt pathway components also were observed. Wnt7a decreased significantly, while Wnt5a, LRP5, β-catenin and c-jun expressions increased. Culture of human articular chondrocytes with or without IL-1 not only induced chondrocyte de-differentiation, but also changed the expression profiles of Wnt components, which suggests that the Wnt pathway is involved in chondrocyte de-differentiation in vitro.

α-Catenin Inhibits β-Catenin-T-cell Factor/Lymphoid Enhancing Factor Transcriptional Activity and Collagen Type II Expression in Articular Chondrocytes through Formation of Gli3R·α-Catenin·β-Catenin Ternary Complex

Chondrocytes, a unique cell type in cartilage tissue, are responsible for the regulation of anabolic and catabolic homeostasis in cartilage-specific extracellular matrix synthesis. Activation of Wnt/β-catenin signaling induces dedifferentiation of articular chondrocytes, resulting in suppression of type II collagen expression. We have shown previously that α-catenin inhibits β-catenin-Tcf/Lef (T-cell factor/lymphoid-enhancing factor) transcriptional activity in articular chondrocytes with a concomitant recovery of type II collagen expression. In the current study, we elucidated the mechanism underlying this inhibition of β-catenin-Tcf/Lef transcriptional activity by α-catenin, showing that it requires direct interaction between α-catenin and β-catenin. We further showed that it involves recruitment of Gli3R, the short transcription-repressing form of the transcription factor Gli3, to β-catenin by α-catenin. The resulting inhibition of β-catenin transcriptional activity leads to increased expression of type II collagen. Gli3R and α-catenin actions are co-dependent: both are necessary for the observed inhibitory effects on β-catenin transcriptional activity. Reducing Gli3R expression levels through activation of Indian Hedgehog (Ihh) signaling also is sufficient to activate β-catenin transcriptional activity, suggesting that the ternary complex, Gli3R·α-catenin·β-catenin, mediates Ihh-dependent activation of Wnt/β-catenin signaling in articular chondrocytes. Collectively, this study shows that α-catenin functions as a nuclear factor that recruits the transcriptional repressor Gli3R to β-catenin to inhibit β-catenin transcriptional activity and dedifferentiation of articular chondrocytes. Finally, osteoarthritic cartilage showed elevated levels of β-catenin and decreased levels of α-catenin and Gli3R, suggesting that decreased levels of α-catenin and Gli3R levels contribute to increased β-catenin transcriptional activity during osteoarthritic cartilage destruction.

Antifibrotic effect of adipose stromalcells on chondrocytes from osteoarthritic patients

PurposesOsteoarthritis (OA) characterised by degeneration of articular cartilage is the most frequent rheumatic disease. Mesenchymal stem cells (MSC) isolated from bone marrow (MSC) or adipose tissues (ASC) secrete a large...

Nicotinamide Phosphoribosyltransferase Is Essential for Interleukin-1β-mediated Dedifferentiation of Articular Chondrocytes via SIRT1 and Extracellular Signal-regulated Kinase (ERK) Complex Signaling

Although much is known about interleukin (IL)-1β and its role as a key mediator of cartilage destruction in osteoarthritis, only limited information is available on IL-1β signaling in chondrocyte dedifferentiation. Here, we have characterized the molecular mechanisms leading to the dedifferentiation of primary cultured articular chondrocytes by IL-1β treatment. IL-1β or lipopolysaccharide, but not phorbol 12-myristate 13-acetate, retinoic acid, or epidermal growth factor, induced nicotinamide phosphoribosyltransferase (NAMPT) expression, showing the association of inflammatory cytokines with NAMPT regulation. SIRT1, in turn, was activated NAMPT-dependently, without any alteration in the expression level. Activation or inhibition of SIRT1 oppositevely regulates IL-1β-mediated chondrocyte dedifferentiation, suggesting this protein as a key regulator of chondrocytes phenotype. SIRT1 activation promotes induction of ERK and p38 kinase activities, but not JNK, in response to IL-1β. Subsequently, ERK and p38 kinase activated by SIRT1 also induce SIRT1 activation, forming a positive feedback loop to sustain downstream signaling of these kinases. Moreover, we found that the SIRT1-ERK complex, but not SIRT1-p38, is engaged in IL-1β-induced chondrocyte dedifferentiation via a Sox-9-mediated mechanism. JNK is activated by IL-1β and modulates dedifferentiation of chondrocytes, but this pathway is independent on NAMPT-SIRT1 signaling. Based on these findings, we propose that IL-1β induces dedifferentiation of articular chondrocytes by up-regulation of SIRT1 activity enhanced by both NAMPT and ERK signaling.

Hyaluronic acid inhibits nitric oxide-induced apoptosis and dedifferentiation of articular chondrocytes in vitro

Nitric oxide is an important mediator in Osteoarthritis (OA), and causes apoptosis and dedifferentiation in articular chondrocytes. Protein kinase Calpha is involved in modulating apoptosis and dedifferentiation of articular chondrocytes induced by nitric oxide. Hyaluronic acid is widely used in the treatment of osteoarthritis and exerts significant chondroprotective effects. The exact mechanisms of its chondroprotective action are not yet fully elucidated. The present study was performed to investigate the effects and mechanisms of hyaluronic acid in NO-induced apoptosis and dedifferentiation of chondrocytes. The ratio of apoptotic cell and cell viability was surveyed by TUNEL, MTT assay and flow cytometry. The expression of aggrecan, type II collagen, and PKCalpha were determined by real-time PCR and Western blot. The expression changes of caspase-3 and bcl-2 was detected by Western blot. The mitochondrial membrane potential (DeltaPsim) was evaluated by Rhodamine-123 fluorescence. HA reduces the TUNEL positive cell, nuclei fragment and the impairment of DeltaPsim. NO-induced chondrocyte dedifferentiation was blocked by HA, which restores expression of aggrecan and type II collagen of chondrocytes and cell viability. HA can block inhibition of PKC-alpha by NO. Our results show that HA blocks NO-induced apoptosis and dedifferentiation of articular chondrocytes by modulation of DeltaPsim and PKCalpha.

Expression of Notch family members in cultured murine articular chondrocytes

The Notch family is involved in cell differentiation during embryogenesis. Osteoarthritic chondrocytes undergo morphological and biochemical changes leading to the de-differentiation process. In the study reported here, we were interested in the involvement of the Notch pathway in murine articular chondrocyte de-differentiation. Articular chondrocytes were subjected to several cell culture passages and treated with or without a Notch inhibitor, N-[N-(3, 5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-Butyl Ester (DAPT). Chondrocyte morphology was studied using optical microscopy. Immunocytochemistry and immunoblot were performed to study the expression of collagens and Notch family members. Without DAPT treatment, chondrocyte de-differentiation resulted in fibroblast-like morphology. This was confirmed by immunocytochemical staining and immunoblot analysis, which showed an increase in collagen type I (col I) and a decrease in collagen type II (col II) expression. With DAPT treatment, de-differentiation was delayed. Immunocytochemistry and immunoblot analysis showed during the first passages inhibition of col II expression, which then was re-instituted during the last passage, suggesting chondrocyte re-differentiation. In the study reported here, we showed that inhibition of the Notch receptor not only delayed the de-differentiation process, but also chondrocyte re-differentiation, which confirms the involvement of the Notch pathway in chondrocyte de-differentiation.

Influence of the growth factors PDGF‐BB, TGF‐β1 and bFGF on the replicative aging of human articular chondrocytes during in vitro expansion

Decreasing replicative potential and dedifferentiation of articular chondrocytes during expansion in cell culture are essential limitations for tissue engineering and cell therapy approaches. Telomeres and telomerase play a key role in cell development, aging, and tumorigenesis. There is evidence that growth factors are involved in regulating telomerase activity. Therefore, the objective was to evaluate the effect of selected growth factors on telomere biology of serially passaged chondrocytes. Human articular chondrocytes were isolated from cartilage of three patients undergoing total knee arthroplasty. The chondrocytes were cultured in monolayer with the growth factors PDGF-BB, TGF-beta1, and bFGF. Telomere length was measured by telomere restriction fragment length assay, and telomerase activity was determined by quantifying the gene expression of its catalytic subunit hTERT by rtPCR. Chondrocytes cultured with PDGF-BB and TGF-beta1 showed a significantly higher proliferation rate than control cells. None of the growth factor cultures revealed an accelerated rate of telomere shortening. Telomerase was not expressed in significant amounts in any of the chondrocyte cultures. Growth factor treatment of chondrocyte cell cultures for cell therapy purposes can be regarded as safe in terms of telomere biology.

Involvement of the notch pathway in the regulation of matrix metalloproteinase 13 and the dedifferentiation of articular chondrocytes in murine cartilage

To demonstrate the activation of the Notch signaling pathway during changes in the phenotype of chondrocytes in vitro, and to assess the influence of Notch on the production of chondrocyte markers. Serial monolayer primary cultures of murine articular chondrocytes (MACs), as a model of chondrocyte dedifferentiation, were prepared. MACs were cultured with or without a Notch inhibitor and transfected with different Notch-expressing vectors. The Notch pathway and chondrocyte marker profiles were assessed by quantitative reverse transcription-polymerase chain reaction, immunoblotting, and immunocytochemistry. Successive passages of MACs resulted in a loss of type II collagen and aggrecan (chondrocyte differentiation markers), an increase in type I collagen (dedifferentiation marker), an increase in Notch ligands, and augmented target gene activity. The Notch inhibitor decreased the type II collagen protein content but had no effect on Col2a1 messenger RNA, while transfection with the constitutive active forms of the Notch1 receptor led to a decrease in type II collagen in transfected cells. In assays to investigate the mechanism of type II collagen breakdown, matrix metalloproteinase 13 (MMP-13) synthesis was regulated in a Notch-dependent manner, whereas MMP-2 synthesis was unchanged. The Notch signaling pathway is associated with decreased type II collagen production during the dedifferentiation of MACs in vitro. This may be correlated with the increase in MMP-13 production linked to activation of Notch.

Ectopic expression of cyclooxygenase-2-induced dedifferentiation in articular chondrocytes

Cyclooxygenase-2 (COX-2) is known to modulate bone metabolism, including bone formation and resorption. Because cartilage serves as a template for endochondral bone formation and because cartilage development is initiated by the differentiation of mesenchymal cells into chondrocytes (Ahrens et al., 1977; Sandell and Adler, 1999; Solursh, 1989), it is of interest to know whether COX-2 expression affect chondrocyte differentiation. Therefore, we investigated the effects of COX-2 protein on differentiation in rabbit articular chondrocyte and chick limb bud mesenchymal cells. Overexpression of COX-2 protein was induced by the COX-2 cDNA transfection. Ectopic expression of COX-2 was sufficient to causes dedifferentiation in articular chondrocytes as determined by the expression of type II collagen via Alcian blue staining and Western blot. Also, COX-2 overexpression caused suppression of SOX-9 expression, a major transcription factor that regulates type II collagen expression, as indicated by the Western blot and RT-PCR. We further examined ectopic expression of COX-2 in chondrifying mesenchymal cells. As expected, COX-2 cDNA transfection blocked cartilage nodule formation as determined by Alcian blue staining. Our results collectively suggest that COX-2 overexpression causes dedifferentiation in articular chondrocytes and inhibits chondrogenic differentiation of mesenchymal cells.