Age-related differences in the accumulation and size of hyaluronan in alginate culture

The alginate bead culture system has unique properties that make it possible to study the accumulation and turnover of macromolecules in two distinct matrix compartments of the cartilage matrix: the cell-associated matrix (CM) and the further removed matrix (FRM). Taking advantage of this culture system, the purpose of this study was to examine age-related changes in the metabolism of hyaluronan (HA) in these two compartments. Bovine chondrocytes, isolated from fetal, young adult, and old adult articular cartilage, were cultured in alginate beads. On Days 7 and 14 of culture, the alginate gel was solubilized, the CM and FRM were separated and macromolecules in both compartments were analyzed. When compared to the cells from fetal and old adult animals, the young adult cells proliferated at the fastest rate. Fetal cells produced a more abundant CM that was richer in proteoglycans (PGs) than the CM of young or old adult cells. With increasing age, there was an increased tendency for PG, collagen, and HA to escape incorporation into the CM and to become immobilized in the FRM. Very striking changes also were observed in the ratio of HA to PG, which increased markedly with age, and in the size of the HA molecules, which decreased markedly with age. The results suggest that the metabolism of HA in cartilage undergoes pronounced age-related changes, some of which are retained during culture in alginate gel. The findings also suggest that the previously documented age-related decrease in the size of HA in native bovine cartilage reflects, at least in part, a biochemical process occurring at the time or at least soon after the glycosaminoglycan chain is synthesized. It does not appear to simply be the result of age-related changes occurring slowly with time after synthesis, as was previously suggested to be the case for human articular cartilage.     

The C5 domain of Col6A3 is cleaved off from the Col6 fibrils immediately after secretion.

In articular cartilage, type VI collagen is concentrated in the pericellular matrix compartment. During protein synthesis and processing at least the alpha3(VI) chain undergoes significant posttranslational modification and cleavage. In this study, we investigated the processing of type VI collagen in articular cartilage. Immunostaining with a specific polyclonal antiserum against the C5 domain of alpha3(VI) showed strong cellular staining seen in nearly all chondrocytes of articular cartilage. Confocal laser-scanning microscopy and immunoelectron microscopy allowed localization of this staining mainly to the cytoplasm and the immediate pericellular matrix. Double-labeling experiments showed a narrow overlap of the C5 domain and the pericellular mature type VI collagen. Our results suggest that at least in human adult articular cartilage the C5 domain of alpha3(VI) collagen is synthesized and initially incorporated into the newly formed type VI collagen fibrils, but immediately after secretion is cut off and is not present in the mature pericellular type VI matrix of articular cartilage.     

The effect of TGF-beta1 and beta-estradiol on glycosaminoglycan and type II collagen distribution in articular chondrocyte cultures

Articular cartilage extracellular matrix (ECM) plays a crucial role in regulating chondrocyte functions via cell-matrix interaction, cytoskeletal organization and integrin-mediated signaling. Factors such as interleukins, basic fibroblast growth factor (bFGF), bone morphogenic proteins (BMPs) and insulin-like growth factor (IGF) have been shown to modulate the synthesis of extracellular matrix in vitro. However, the effects of TGF-beta1 and beta-estradiol in ECM regulation require further investigation, although there have been suggestions that these factors do play a positive role. To establish the role of these factors on chondrocytes derived from articular joints, a study was conducted to investigate the effects of TGF-beta1 and beta-estradiol on glycosaminoglycan secretion and type II collagen distribution (two major component of cartilage ECM in vivo). Thus, chondrocyte cultures initiated from rabbit articular cartilage were treated with 10ng/ml of TGF-beta1, 10nM of beta-estradiol or with a combination of both factors. Sulphated glycosaminoglycan (GAG) and type II collagen levels were then measured in both these culture systems. The results revealed that the synthesis of GAG and type II collagen was shown to be enhanced in the TGF-beta1 treated cultures. This increase was also noted when TGF-beta1 and beta-estradiol were both used as culture supplements. However, beta-estradiol alone did not appear to affect GAG or type II collagen deposition. There was also no difference between the amount of collagen type II and GAG being expressed when chondrocyte cultures were treated with TGF-beta1 when compared with cultures treated with combined factors. From this, we conclude that although TGF-beta1 appears to stimulate chondrocyte ECM synthesis, beta-estradiol fails to produce similar effects. The findings of this study confirm that contrary to previous claims, beta-estradiol has little or no effect on chondrocyte ECM synthesis. Furthermore, the use of TGF-beta1 may be useful in future studies looking into biological mechanisms by which ECM synthesis in chondrocyte cultures can be augmented, particularly for clinical application.     

Extracellular matrix changes in knee joint cartilage following bone-active drug treatment

Certain drugs or treatments that are known to affect bone quality or integrity might have side effects on the extracellular matrix of articular cartilage. We investigated the effects of vitamin D and calcium deficiency, estrogen deficiency, and hypercortisolism alone or in combination with bisphosphonates or sodium fluoride in an animal model, viz., the Göttingen miniature pig (n=29). The articular cartilage from knee joints was analyzed for its content of glycosaminoglycans (GAGs, as macromolecules responsible for the elasticity of articular cartilage) by a spectrometric method with dimethylene blue chloride. In cryo- or paraffin sections, alkaline phosphatase (AP, as an enzyme indicating mineralization or reorganization of articular cartilage matrix) was localized by enzyme histochemistry, and positive cells were counted, whereas differently sulfated GAGs were stained histochemically. A significant decrease in GAG content was measured in ovariectomized and long-term glucocorticoid-treated animals compared with untreated animals. In the glucocorticoid/sodium fluoride group, GAGs were significantly diminished, and significantly fewer AP-positive chondrocytes were counted compared with the control. GAG content was slightly higher, and significantly more AP-positive chondrocytes were counted in short-term glucocorticoid-treated animals then in the control group. GAGs, as part of proteoglycans, are responsible for the water-storage capacity that gives articular cartilage its unique property of elasticity. Thus, ovariectomy and long-term glucocorticoid therapy, especially when combined with sodium fluoride, have detrimental effects on this tissue.This work was in part supported by Deutsche Forschungsgemeinschaft (DFG) project no. Schr 430/5–1, 5–2 and G 1289/1–1, 1–2     

Effects of rapid cooling on articular cartilage

In order to improve the technique and protocols of cryopreservation of articular cartilage, a study was carried out to assess the effects of rapid cooling on the intact articular cartilage. Cartilage slices with a thickness ranging from 0.2 to 0.5 mm taken from bovine metacarpal-phalangeal joints were subjected to rapid cooling by immersing them in liquid nitrogen with and without treatment of the VS55 cryoprotective agent (CPA). The ultrastructure, chondrocyte viability, swelling property, and glycosaminoglycan (GAG) content were then examined before and after cryopreservation to give qualitative and quantitative evaluation on the functional state of both chondrocytes and extracellular matrix. The transmission electron microscopy study demonstrated that damage to chondrocytes without CPA was far more pronounced than those with VS55 protection while the structure of the extracellular matrix altered little in either group. The cell viability assay showed that although the exposure to VS55 led to about 36% chondrocytes losing membrane integrity, the VS55 could provide protection to chondrocytes during rapid cooling and thawing, with approximately 51% of the cells having survived rapid cooling compared to fewer than 5% in the absence of CPA. There were no significant differences in degrees of swelling or the GAG contents of cartilage slices after cryopreservation indicating rapid freezing caused little damage to the matrix. Future research activities include searching improved CPA formulation, optimising the treatment protocol and investigating the long-term effects of rapid cooling on articular cartilage.     

Synoviocyte Derived-Extracellular Matrix Enhances Human Articular Chondrocyte Proliferation and Maintains Re-Differentiation Capacity at Both Low and Atmospheric Oxygen Tensions

BackgroundCurrent tissue engineering methods are insufficient for total joint resurfacing, and chondrocytes undergo de-differentiation when expanded on tissue culture plastic. De-differentiated chondrocytes show poor re-differentiation in culture, giving reduced glycosaminoglycan (GAG) and collagen matrix accumulation. To address this, porcine synoviocyte-derived extracellular matrix and low (5%) oxygen tension were assessed for their ability to enhance human articular chondrocyte expansion and maintain re-differentiation potential.MethodsPorcine synoviocyte matrices were devitalized using 3 non-detergent methods. These devitalized synoviocyte matrices were compared against tissue culture plastic for their ability to support human chondrocyte expansion. Expansion was further compared at both low (5%), and atmospheric (20%) oxygen tension on all surfaces. Expanded cells then underwent chondrogenic re-differentiation in aggregate culture at both low and atmospheric oxygen tension. Aggregates were assessed for their GAG and collagen content both biochemically and histologically.ResultsHuman chondrocytes expanded twice as fast on devitalized synoviocyte matrix vs. tissue culture plastic, and cells retained their re-differentiation capacity for twice the number of population doublings. There was no significant difference in growth rate between low and atmospheric oxygen tension. There was significantly less collagen type I, collagen type II, aggrecan and more MMP13 expression in cells expanded on synoviocyte matrix vs. tissue culture plastic. There were also significant effects due to oxygen tension on gene expression, wherein there was greater collagen type I, collagen type II, SOX9 and less MMP13 expression on tissue culture plastic compared to synoviocyte matrix. There was a significant increase in GAG, but not collagen, accumulation in chondrocyte aggregates re-differentiated at low oxygen tension over that achieved in atmospheric oxygen conditions.ConclusionsSynoviocyte-derived matrix supports enhanced expansion of human chondrocytes such that the chondrocytes are maintained in a state from which they can re-differentiate into a cartilage phenotype after significantly more population doublings. Also, low oxygen tension supports GAG, but not collagen, accumulation. These findings are a step towards the production of a more functional, tissue engineered cartilage.     

Accumulation of advanced glycation end products decreases collagen turnover by bovine chondrocytes

The integrity of the collagen network is essential for articular cartilage to fulfill its function in load support and distribution. Damage to the collagen network is one of the first characteristics of osteoarthritis. Since extensive collagen damage is considered irreversible, it is crucial that chondrocytes maintain a functional collagen network. We investigated the effects of advanced glycation end products (AGEs) on the turnover of collagen by articular cartilage chondrocytes. Increased AGE levels (by culturing in the presence of ribose) resulted in decreased collagen synthesis (P < 0.05) and decreased MMP-mediated collagen degradation (P < 0.02). The latter could be attributed to increased resistance of the collagen network to MMPs (P < 0.05) as well as the decreased production of MMPs by chondrocytes (P < 0.02). Turnover of a protein is determined by its synthesis and degradation rates and therefore these data indicate that collagen turnover is decreased at enhanced AGE levels. Since AGE levels in human cartilage increase approximately 50 fold between age 20 and 80, cartilage collagen turnover likely decreases with increasing age. Impaired collagen turnover adversely affects the capacity of chondrocytes to remodel and/or repair its extracellular matrix. Consequently, age-related accumulation of AGE (via decreased collagen turnover) may contribute to the development of cartilage damage in osteoarthritis.     

The influence of oxygen and hydrostatic pressure on articular chondrocytes and adherent bone marrow cells in vitro

Tissue engineering of articular cartilage from chondrocytes or stem cells is considered to be a potential aspect in the treatment of cartilage defects. In order to optimize culture conditions the influence of low oxygen tension (5%) - single or in combination with intermittent hydrostatic pressure (HP: 30/2 min on/off loading; 0.2 MPa) - on the biosynthetic activity (sulfate and proline incorporation) of human osteoarthritic chondrocytes cultured on collagen I/III membranes was investigated. Additionally, chondrogenesis from high density or monolayer cultures of bovine adherent bone marrow cells (aBMC) with and without chondrogenic medium supplements (CM) was analyzed by RT-PCR (mRNA expression of aggrecan and collagen type II). We could show that low oxygen tension increases significantly the biosynthesis of collagen I/III membrane-associated chondrocytes and even higher under co-stimulation with HP. While there is no chondrogenesis in monolayer cultures, CM induces expression of cartilage matrix molecules in high density cultures of aBMC which is even increased under the influence of low oxygen tension. Both, low oxygen tension and HP without CM are alone not sufficient stimuli for chondrogenesis. It can be concluded that low oxygen tension and HP might be useful tools in cartilage tissue engineering and that these physico-chemical factors promote but do not induce chondrogenesis under the given conditions.     

Synthesis rates and binding kinetics of matrix products in engineered cartilage constructs using chondrocyte-seeded agarose gels

Large-sized cartilage constructs suffer from inhomogeneous extracellular matrix deposition due to insufficient nutrient availability. Computational models of nutrient consumption and tissue growth can be utilized as an efficient alternative to experimental trials to optimize the culture of large constructs; models require system-specific growth and consumption parameters. To inform models of the [bovine chondrocyte]−[agarose gel] system, total synthesis rate (matrix accumulation rate+matrix release rate) and matrix retention fractions of glycosaminoglycans (GAG), collagen, and cartilage oligomeric matrix protein (COMP) were measured either in the presence (continuous or transient) or absence of TGF-β3 supplementation. TGF-β3's influences on pyridinoline content and mechanical properties were also measured. Reversible binding kinetic parameters were characterized using computational models. Based on our recent nutrient supplementation work, we measured glucose consumption and critical glucose concentration for tissue growth to computationally simulate the culture of a human patella-sized tissue construct, reproducing the experiment of Hung et al. (2003). Transient TGF-β3 produced the highest GAG synthesis rate, highest GAG retention ratio, and the highest binding affinity; collagen synthesis was elevated in TGF-β3 supplementation groups over control, with the highest binding affinity observed in the transient supplementation group; both COMP synthesis and retention were lower than those for GAG and collagen. These results informed the modeling of GAG deposition within a large patella construct; this computational example was similar to the previous experimental results without further adjustments to modeling parameters. These results suggest that these nutrient consumption and matrix synthesis models are an attractive alternative for optimizing the culture of large-sized constructs.     

Effect of extracellular ph on matrix synthesis by chondrocytes in 3D agarose gel

In cartilage tissue engineering, the determination of the most appropriate cell/tissue culture conditions to maximize extracellular matrix synthesis is of major importance. The extracellular pH plays an important role in affecting energy metabolism and matrix synthesis by chondrocytes. In this study, chondrocytes were isolated from bovine articular cartilage, embedded in agarose gel, and cultured at varied pH levels (7.3-6.6). Rate of lactate production, total glycosaminoglycan (GAG) and collagen synthesis, as well as total cell numbers and cell viability were evaluated after culturing for up to 7 days. The results showed the rate of lactic acid production over the 7-day culture was significantly affected by extracellular pH; acidic pH markedly inhibited the production of lactate. Also, a biphasic response to extracellular pH in regard to total GAG synthesis was observed; the maximum synthesis was seen at pH 7.2. However, the collagen synthesis was not pH-dependent within the pH range explored. In addition, within the conditions studied, total cell numbers and cell viability were not significantly affected by extracellular pH. In conclusion, even minor changes in extracellular pH could markedly affect the metabolic activities and biosynthetic ability of chondrocytes. Consequently, the control of extracellular pH condition is crucially important for successful cartilage tissue engineering and for the study of chondrocyte physiology and functions.     

Structured three-dimensional co-culture of mesenchymal stem cells with meniscus cells promotes meniscal phenotype without hypertrophy

Menisci play a crucial role in weight distribution, load bearing, shock absorption, lubrication, and nutrition of articular cartilage within the knee joint. Damage to the meniscus typically does not heal spontaneously due to its partial avascular nature. Partial or complete meniscectomy is a common clinical treatment of the defective meniscus. However, this procedure ultimately leads to osteoarthritis due to increased mechanical stress to the articular cartilage. Meniscus tissue engineering offers a promising solution for partial or complete meniscus deficiency. Mesenchymal stem cells (MSC) have the potential to differentiate into meniscal fibrochondrocyte as well as deliver trophic effects to the differentiated cells. This study tested the feasibility of using MSC co-cultured with mature meniscal cells (MC) for meniscus tissue engineering. Structured cell pellets were created using MC and MSC at varying ratios (100:0, 75:25, 50:50, 25:75, and 0:100) and cultured with or without transforming growth factor-beta 3 supplemented chondrogenic media for 21 days. The meniscal and hypertrophic gene expression, gross appearance and structure of the pellets, meniscus extracellular matrix (ECM), histology and immunohistochemistry of proteoglycan and collagen were evaluated. Co-culture of MC with MSC at 75:25 demonstrated highest levels of collagen type I and glycosaminoglycans (GAG) production, as well as the lowest levels of hypertrophic genes, such as COL10A1 and MMP13. All co-culture conditions showed better meniscus ECM production and hypertrophic inhibition as compared to MSC culture alone. The collagen fiber bundles observed in the co-cultures are important to produce heterogenic ECM structure of meniscus. In conclusion, co-culturing MC and MSC is a feasible and efficient approach to engineer meniscus tissue with enhanced ECM production without hypertrophy.     

Redifferentiation of chondrocytes and cartilage formation under intermittent hydrostatic pressure

Since articular cartilage is subjected to varying loads in vivo and undergoes cyclic hydrostatic pressure during periods of loading, it is hypothesized that mimicking these in vivo conditions can enhance synthesis of important matrix components during cultivation in vitro. Thus, the influence of intermittent loading during redifferentiation of chondrocytes in alginate beads, and during cartilage formation was investigated. A statistically significant increased synthesis of glycosaminoglycan and collagen type II during redifferentiation of chondrocytes embedded in alginate beads, as well as an increase in glycosaminoglycan content of tissue-engineered cartilage, was found compared to control without load. Immunohistological staining indicated qualitatively a high expression of collagen type II for both cases.     

Competing regulation of matrix biosynthesis by mechanical and IGF-1 signalling in elderly human articular cartilage in vitro

This study investigates the separate and combined effects of IGF-1 and mechanical loads on chondrocytes in elderly human femoral head articular cartilage. Full depth biopsies of articular cartilage were subjected to either no load, static or cyclic (2 s on/2 s off) loading in unconfined compression at a stress of 1 MPa for 48 h with or without IGF-1 (300 ng ml(-1)). Chondrocyte biosynthetic activity was measured using 35S-sulphate and 3H-leucine during the last 24 h of loading. IGF-1 alone increased the rates of isotope incorporation, by 80% for 35S-SO4 and 40% for 3H-leucine, whereas loading alone reduced matrix biosynthesis. Applying load (cyclic or static) in the presence of IGF-1 returned the incorporation rates to their unstimulated levels. This study suggests elderly human articular cartilage is responsive to stimulation by IGF-1 but mechanical factors seem to act sufficiently strongly in the opposite direction to cancel this response.     

Importance of collagen orientation and depth-dependent fixed charge densities of cartilage on mechanical behavior of chondrocytes

The collagen network and proteoglycan matrix of articular cartilage are thought to play an important role in controlling the stresses and strains in and around chondrocytes, in regulating the biosynthesis of the solid matrix, and consequently in maintaining the health of diarthrodial joints. Understanding the detailed effects of the mechanical environment of chondrocytes on cell behavior is therefore essential for the study of the development, adaptation, and degeneration of articular cartilage. Recent progress in macroscopic models has improved our understanding of depth-dependent properties of cartilage. However, none of the previous works considered the effect of realistic collagen orientation or depth-dependent negative charges in microscopic models of chondrocyte mechanics. The aim of this study was to investigate the effects of the collagen network and fixed charge densities of cartilage on the mechanical environment of the chondrocytes in a depth-dependent manner. We developed an anisotropic, inhomogeneous, microstructural fibril-reinforced finite element model of articular cartilage for application in unconfined compression. The model consisted of the extracellular matrix and chondrocytes located in the superficial, middle, and deep zones. Chondrocytes were surrounded by a pericellular matrix and were assumed spherical prior to tissue swelling and load application. Material properties of the chondrocytes, pericellular matrix, and extracellular matrix were obtained from the literature. The loading protocol included a free swelling step followed by a stress-relaxation step. Results from traditional isotropic and transversely isotropic biphasic models were used for comparison with predictions from the current model. In the superficial zone, cell shapes changed from rounded to elliptic after free swelling. The stresses and strains as well as fluid flow in cells were greatly affected by the modulus of the collagen network. The fixed charge density of the chondrocytes, pericellular matrix, and extracellular matrix primarily affected the aspect ratios (height/width) and the solid matrix stresses of cells. The mechanical responses of the cells were strongly location and time dependent. The current model highlights that the collagen orientation and the depth-dependent negative fixed charge densities of articular cartilage have a great effect in modulating the mechanical environment in the vicinity of chondrocytes, and it provides an important improvement over earlier models in describing the possible pathways from loading of articular cartilage to the mechanical and biological responses of chondrocytes.     

Extent of cell differentiation and capacity for cartilage synthesis in human adult adipose‐derived stem cells: Comparison with fetal chondrocytes

This study evaluated the extent of differentiation and cartilage biosynthetic capacity of human adult adipose‐derived stem cells relative to human fetal chondrocytes. Both types of cell were seeded into nonwoven‐mesh polyglycolic acid (PGA) scaffolds and cultured under dynamic conditions with and without addition of TGF‐β1 and insulin. Gene expression for aggrecan and collagen type II was upregulated in the stem cells in the presence of growth factors, and key components of articular cartilage such as glycosaminoglycan (GAG) and collagen type II were synthesized in cultured tissue constructs. However, on a per cell basis and in the presence of growth factors, accumulation of GAG and collagen type II were, respectively, 3.4‐ and 6.1‐fold lower in the stem cell cultures than in the chondrocyte cultures. Although the stem cells synthesized significantly higher levels of total collagen than the chondrocytes, only about 2.4% of this collagen was collagen type II. Relative to cultures without added growth factors, treatment of the stem cells with TGF‐β1 and insulin resulted in a 59% increase in GAG synthesis, but there was no significant change in collagen production even though collagen type II gene expression was upregulated 530‐fold. In contrast, in the chondrocyte cultures, synthesis of collagen type II and levels of collagen type II as a percentage of total collagen more than doubled after growth factors were applied. Although considerable progress has been achieved to develop differentiation strategies and scaffold‐based culture techniques for adult mesenchymal stem cells, the extent of differentiation of human adipose‐derived stem cells in this study and their capacity for cartilage synthesis fell considerably short of those of fetal chondrocytes. Biotechnol. Bioeng. 2010;107: 393–401. © 2010 Wiley Periodicals, Inc.     

Differing in vitro biology of equine,ovine, porcine and human articular chondrocytes derived from the knee joint: an immunomorphological study

For lack of sufficient human cartilage donors, chondrocytes isolated from various animal species are used for cartilage tissue engineering. The present study was undertaken to compare key features of cultured large animal and human articular chondrocytes of the knee joint. Primary chondrocytes were isolated from human, porcine, ovine and equine full thickness knee joint cartilage and investigated flow cytometrically for their proliferation rate. Synthesis of extracellular matrix proteins collagen type II, cartilage proteoglycans, collagen type I, fibronectin and cytoskeletal organization were studied in freshly isolated or passaged chondrocytes using immunohistochemistry and western blotting. Chondrocytes morphology, proliferation, extracellular matrix synthesis and cytoskeleton assembly differed substantially between these species. Proliferation was higher in animal derived compared with human chondrocytes. All chondrocytes expressed a cartilage-specific extracellular matrix. However, after monolayer expansion, cartilage proteoglycan expression was barely detectable in equine chondrocytes whereby fibronectin and collagen type I deposition increased compared with porcine and human chondrocytes. Animal-derived chondrocytes developed more F-actin fibers during culturing than human chondrocytes. With respect to proliferation and extracellular matrix synthesis, human chondrocytes shared more similarity with porcine than with ovine or equine chondrocytes. These interspecies differences in chondrocytes in vitro biology should be considered when using animal models.     

FAK mediates signal crosstalk between type II collagen and TGF-beta 1 cascades in chondrocytic cells

The purpose of this study was to evaluate the mechanism of crosstalk between the type II collagen and TGF-β1 signaling pathways in chondrocytic cells. Articular chondrocytes, isolated from porcine knee cartilage, and the SW1353 cell line were cultured on either type II collagen-coated or -uncoated plates in the presence or absence of TGF-β1. Expression of pSMAD 2, pSMAD 3, pFAK_Y397 and pFAK_Y925 in articular chondrocytes and the SW1353 cell line was analyzed by immunoblotting. Cell proliferation rates and glycosaminoglycan (GAG) content was determined after treatment with type II collagen or/and TGF-β1. For inhibition study, human FAK-specific RNA small interference (siFAK) in SW1353 cell line was performed. In this study, expression of pSMAD 2, pSMAD 3, pFAK_Y397 and pFAK_Y925 were synergistically increased by co-treatment with type II collagen and TGF-β1 in articular chondrocytes. The proliferation of porcine articular chondrocytes and GAG secretion in SW1353 cells were synergistically increased by co-stimulation with type II collagen and TGF-β1. Synergistically increased expression and nuclear translocation of pSMAD 2 and pSMAD 3 and GAG secretion of SW1353 cells were significantly inhibited by siFAK transfection. Therefore, we suggest that FAK-SMAD 2/3 mediates signal crosstalk between type II collagen and TGF-β1 and regulates GAG secretion in chondrocytic cells.     

Chondrocyte extracellular matrix synthesis and turnover are influenced by static compression in a new alginate disk culture system

The goal of this study was to examine the effects of mechanical compression on chondrocyte biosynthesis of extracellular matrix (ECM) components during culture in a new alginate disk culture system. Specifically, we have examined chondrocyte biosynthesis rates, and the structure of aggrecan core protein species present in the cell-associated matrix (CM), in the further removed matrix (FRM) and in the surrounding culture medium. In this alginate disk culture system, chondrocytes can be subjected to mechanical deformations similar to those experienced in vivo. Our results show that over an 8-week culture period, chondrocytes synthesize a functional ECM and can respond to mechanical forces similarly to chondrocytes maintained in native cartilage. In the alginate disk system, static compression was shown to decrease and dynamic compression to increase synthesis of aggrecan of bovine chondrocytes. Western blot analysis of the core proteins of aggrecan molecules identified a number of different species that were present in different relative amounts in the CM, FRM, and medium. Over 21 days of culture, the predominant form of aggrecan found in the ECM was a full-length link-stabilized species. In addition, our data show that the application of 40 h of static compression caused an increase in the proportion of newly synthesized aggrecan molecules released into the medium. However, this was not accompanied by a significant change in the size and composition of aggrecan and aggrecan fragments in the different compartments, suggesting that mechanical compression did not alter the catabolic pathways. Together, these data show that chondrocyte function is maintained in an alginate disk culture system and that this culture system is a useful model to examine chondrocyte ECM assembly and some aspects of catabolism normally found in vivo.     

Potential involvement of oxidative stress in cartilage senescence and development of osteoarthritis: oxidative stress induces chondrocyte telomere instability and downregulation of chondrocyte function

Oxidative stress leads to increased risk for osteoarthritis (OA) but the precise mechanism remains unclear. We undertook this study to clarify the impact of oxidative stress on the progression of OA from the viewpoint of oxygen free radical induced genomic instability, including telomere instability and resulting replicative senescence and dysfunction in human chondrocytes. Human chondrocytes and articular cartilage explants were isolated from knee joints of patients undergoing arthroplastic knee surgery for OA. Oxidative damage and antioxidative capacity in OA cartilage were investigated in donor-matched pairs of intact and degenerated regions of tissue isolated from the same cartilage explants. The results were histologically confirmed by immunohistochemistry for nitrotyrosine, which is considered to be a maker of oxidative damage. Under treatment with reactive oxygen species (ROS; 0.1 μmol/l H₂O₂) or an antioxidative agent (ascorbic acid: 100.0 μmol/l), cellular replicative potential, telomere instability and production of glycosaminoglycan (GAG) were assessed in cultured chondrocytes. In tissue cultures of articular cartilage explants, the presence of oxidative damage, chondrocyte telomere length and loss of GAG to the medium were analyzed in the presence or absence of ROS or ascorbic acid. Lower antioxidative capacity and stronger staining of nitrotyrosine were observed in the degenerating regions of OA cartilages as compared with the intact regions from same explants. Immunostaining for nitrotyrosine correlated with the severity of histological changes to OA cartilage, suggesting a correlation between oxidative damage and articular cartilage degeneration. During continuous culture of chondrocytes, telomere length, replicative capacity and GAG production were decreased by treatment with ROS. In contrast, treatment with an antioxidative agent resulted in a tendency to elongate telomere length and replicative lifespan in cultured chondrocytes. In tissue cultures of cartilage explants, nitrotyrosine staining, chondrocyte telomere length and GAG remaining in the cartilage tissue were lower in ROS-treated cartilages than in control groups, whereas the antioxidative agent treated group exhibited a tendency to maintain the chondrocyte telomere length and proteoglycan remaining in the cartilage explants, suggesting that oxidative stress induces chondrocyte telomere instability and catabolic changes in cartilage matrix structure and composition. Our findings clearly show that the presence of oxidative stress induces telomere genomic instability, replicative senescence and dysfunction of chondrocytes in OA cartilage, suggesting that oxidative stress, leading to chondrocyte senescence and cartilage ageing, might be responsible for the development of OA. New efforts to prevent the development and progression of OA may include strategies and interventions aimed at reducing oxidative damage in articular cartilage.