Chondrocyte Proteoglycan Synthesis Research Articles

Effects of sesamin on chondroitin sulfate proteoglycan synthesis induced by interleukin-1beta in human chondrocytes

BackgroundNumerous studies have reported on the health benefits of sesamin, a major lignin found in sesame (S. indicum) seeds. Recently, sesamin was shown to have the ability to promote chondroitin sulfate proteoglycan synthesis in normal human chondrocytes. This study assesses the anti-inflammatory effect of sesamin on proteoglycans production in 3D chondrocyte cultures.MethodsTo evaluate the effects of sesamin on IL-1β-treated human articular chondrocytes (HAC) pellets, the pellets were pre-treated with IL-1β then cultured in the presence of various concentrations of sesamin for 21 days. During that period, the expression of IL-1β, glycosaminoglycans (GAGs) content and Chondroitin sulfate proteoglycans (CSPGs) synthesis genes (ACAN, XT-1, XT-2, CHSY1 and ChPF) was measured. The GAGs accumulation in the extracellular matrix was determined on day 21 by histological analysis.ResultsThere was clear evidence that sesamin upregulated expression of all the CSPGs synthesis genes, in contrast to the down-regulation of IL-1β expression both in genes and in protein levels. The level of release and matrix accumulation of GAGs in IL-1β pre-treated HAC pellets in the presence of sesamin was recovered. These results correlate with the histological examination which showed that sesamin enhanced matrix CSPGs accumulation.ConclusionsSesamin enhances CSPGs synthesis, suppresses IL-1β expression and ameliorates IL-1β induced inflammation in human chondrocytes. Sesamin could have therapeutic benefits for treating inflammation in osteoarthritis.

Pivotal role of Sirt6 in the crosstalk among ageing, metabolic syndrome and osteoarthritis

Osteoarthritis (OA) is a chronic degenerative joint disorder commonly associated with metabolic syndrome. As ageing and obesity has a great impact on the initiation/severity of OA, herein we sought to investigate the involvement of Sirt6 in the crosstalk between ageing and metabolic syndrome/OA. Sirt6 haploinsufficiency in mice promoted the expression of inflammatory cytokines in the IPFP. Enhanced inflammation of the IPFP in the aged Sirt6 ± HFD group was paralleled with accelerated OA change, including osteophyte growth and chondrocyte hypertrophy. Conversely, mesenchyme-specific Sirt6-deficient mice revealed both attenuated chondrocyte hypertrophy and proteoglycan synthesis, although chondrocyte senescence was enhanced as shown in the aged WT mice. Thus Sirt6 has key roles in the relationship among ageing, metabolic syndrome, and OA.

Evidence for an Important Role of Smad-7 in Intervertebral Disc Degeneration.

Smad-7 inhibited the transforming growth factor beta (TGF-β)-induced proteoglycan synthesis in chondrocytes and completely antagonized the effect of TGF-β on the proliferation of the cells. The aim of this study was to evaluate the contribution of Smad-7 to the pathophysiology of disc degeneration by determining the expression of Smad-7 in the degenerative intervertebral discs and its effect on the extracellular matrix metabolism of disc cells. Instability of the lumbar spine produced by imbalanced dynamic and static forces was used to induce intervertebral disc degeneration in rats. The expression of Smad-7 was assessed by the immunohistochemical method. Disc cell apoptosis was detected by in situ TUNEL staining. The effect of Smad-7 overexpression on the matrix metabolism of disc cells was analyzed in vitro by real-time polymerase chain reaction (PCR) and Western blotting. Finally, intradiscal injection of the Smad-7 overexpression lentivirus was performed to evaluate the in vivo effect of Smad-7 on disc degeneration. Radiographic and histomorphological examinations showed that lumbar disc degeneration became more and more severe in the rats with induced instability. Immunohistochemical observation demonstrated increasing protein expression of Smad-7 in the degenerative discs. A significantly positive correlation was found between Smad-7 expression and the degree of disc degeneration and between Smad-7 expression and disc cell apoptosis. Overexpression of Smad-7 in disc cells inhibited the expression of TGF-β1, collagen type-I, collagen type-II, and aggrecan and promoted the expression of MMP-13, but did not change the expression of ADAMTS-5. The in vivo findings illustrated that intradiscal injection of lentivirus vector with Smad-7 overexpression accelerated the progress of disc degeneration. In conclusion, Smad-7 was highly expressed in the degenerative discs. Overexpression of Smad-7 weakened the protective role of TGF-β and accelerated the progress of disc degeneration. Interference on Smad-7 might be a potential therapeutic method for the prevention and treatment of degenerative disc diseases.

UDP-glucose dehydrogenase modulates proteoglycan synthesis in articular chondrocytes: its possible involvement and regulation in osteoarthritis.

IntroductionThe objective of this study was to investigate the possible role of UDP-glucose dehydrogenase (UGDH) in osteoarthritis (OA) and uncover whether, furthermore how interleukin-1beta (IL-1β) affects UGDH gene expression.MethodsUGDH specific siRNAs were applied to determine the role of UGDH in proteoglycan (PG) synthesis in human articular chondrocytes. Protein levels of UGDH and Sp1 in human and rat OA cartilage were detected. Then, human primary chondrocytes were treated with IL-1β to find out whether and how IL-1β could regulate the gene expression of UGDH and its trans-regulators, that is Sp1, Sp3 and c-Krox. Finally, p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 and stress-activated protein kinase/c-Jun N-terminal kinase (SAP/JNK) inhibitor SP600125 were used to pick out the pathway that mediated the IL-1β-modulated PGs synthesis and gene expression of UGDH, Sp1, Sp3 and c-Krox.ResultsUGDH specific siRNAs markedly inhibited UGDH mRNA and protein expression, and thus led to an obvious suppression of PGs synthesis in human articular chondrocytes. UGDH protein level in human and rat OA cartilage were much lower than the corresponding controls and negatively correlated to the degree of OA. Decrease in Sp1 protein level was also observed in human and rat OA cartilage respectively. Meanwhile, IL-1β suppressed UGDH gene expression in human articular chondrocytes in the late phase, which also modulated gene expression of Sp1, Sp3 and c-Krox and increased both Sp3/Sp1 and c-Krox/Sp1 ratio. Moreover, the inhibition of SAP/JNK and p38 MAPK pathways both resulted in an obvious attenuation of the IL-1β-induced suppression on the UGDH gene expression.ConclusionsUGDH is essential in the PGs synthesis of articular chondrocytes, while the suppressed expression of UGDH might probably be involved in advanced OA, partly due to the modulation of p38 MAPK and SAP/JNK pathways and its trans-regulators by IL-1β.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-014-0484-2) contains supplementary material, which is available to authorized users.

Smad4 regulates growth plate chondrocyte proliferation, columnar organization and proteoglycan synthesis

Searchable abstracts of presentations at key conferences on calcified tissues ISSN 2052-1219 (online)

Extracellular nicotinamide phosphoribosyltransferase (NAMPT/visfatin) inhibits insulin-like growth factor-1 signaling and proteoglycan synthesis in human articular chondrocytes

IntroductionObesity is one of the major risk factors for the development of osteoarthritis (OA). Although the mechanical factors appear to be critical, recent studies have suggested a role for adipokines in cartilage degradation. Chondrocytes from osteoarthritic cartilage respond poorly to insulin-like growth factor-1 (IGF-1) and the molecular mechanism(s) involved is not clearly understood. The purpose of the present study was to determine the role of extracellular nicotinamide phosphoribosyltransferase (eNAMPT/visfatin), a newly described adipokine, in regulating IGF-1 function in chondrocytes.MethodsHuman articular chondrocytes isolated from normal ankle cartilage were pretreated with eNAMPT (0.1 to 5.0 μg/ml) overnight followed by stimulation with IGF-1 (50 ng/ml) for 24 hours, and proteoglycan synthesis was measured by [35S]sulfate incorporation. Chondrocytes were pretreated with eNAMPT overnight followed by IGF-1 for 10 minutes, and the cell lysates were immunoblotted for various signaling proteins that are activated by IGF-1 using phosphospecific antibodies. In addition, chondrocytes were pretreated with mitogen-activated protein kinase kinase inhibitor (U0126) prior to stimulation with eNAMPT and IGF-1.ResultsPretreatment of chondrocytes with eNAMPT inhibited IGF-1-stimulated proteoglycan synthesis in a dose-dependent manner. Treatment of chondrocytes with eNAMPT inhibited IGF-1-induced phosphorylation of signaling molecules, including insulin receptor substrate-1 and AKT. Interestingly, pretreatment of chondrocytes with eNAMPT did not inhibit IGF-1-mediated phosphorylation of the IGF-1 receptor; however, it stimulated a sustained phosphorylation of the extracellular signal-regulated kinase (ERK)/mitogen activated protein kinase (MAPK) signaling pathway. Inhibition of the ERK/MAPK signaling pathway restored IGF-1-mediated insulin receptor substrate-1 and AKT phosphorylation.ConclusionsOur study demonstrates that eNAMPT/visfatin inhibits IGF-1 function in articular chondrocytes by activating the ERK/MAPK pathway independent of the IGF-1 receptor. Since eNAMPT levels are elevated in the synovial fluid of OA patients, the signaling pathway activated by eNAMPT could contribute to IGF-1 resistance in OA.

Untersuchung der Toxizität von Polyvinyl-Pyrrolidon-Jod an fibroblastären Zellen (BALB-3T3) und primären humanen Knorpelzellen

Povidone-iodine (polyvinyl-pyrrolidone-iodine complex, PVP-iodine, CAS 25655-41-8) is a commonly used antiseptic because of its broad spectrum of antimicrobial effect and its comparatively low allergic risk. It is also used for open joint lavage. Animal and organ culture studies provide controversial results about the risk of cartilage damage due to povidone-iodine. There is a paucity of in vitro study data concerning the effect of povidone-iodine on chondrocyte cultures are still missing. The aim of this study was therefore to investigate the effect of different concentrations and exposition times of povidone-iodine on cell growth and differentiation of human chondrocytes. Using of a vitality test (MTT) and a proliferation assay (BrdU) in the fibroblast-like cell line BALB3T3, suitable concentrations and incubation times were identified to investigate the influence of povidone-iodine on proteoglycan synthesis and DNA synthesis of primary human chondrocytes. Concentrations of up to 1% povidone-iodine had no significant effect on proteoglycan and DNA synthesis of chondrocytes after incubation for 30 min. An incubation time of 24 h did not inhibit DNA- and proteoglycan synthesis, until a concentration of 0.2% povidone-iodine was used. DNA synthesis rate was impaired after 10 min incubation with 0.2% and fully inhibited with 1% povidone iodine. BALB3T3 reacted more sensitively than chondrocytes. Vitality and proliferation rate were fully inhibited at a concentration of 0.5% after the same exposition time. However, cells recovered 24 h after 30 min incubation with 0.5% povidone-iodine. After incubation with 5% povidone iodine cells did not recover. From the results it can be concluded that low concentrations of povidone-iodine (< 1%) and short incubation times (< 30 min) have no damaging influence on chondrocytes. Previous studies have reported the antimicrobial effectiveness of low concentrations of povidone-iodine on the reduction of tissue damage by microorganisms. Data from previus studies and the current findings from this investigation support the clinical use of povidone-iodine at low concentrations and short incubation times for antiseptic treatment of cartilage tissues.

CCN3-mediated promotion of sulfated proteoglycan synthesis in rat chondrocytes from developing joint heads.

Chondrocytes forming articular cartilage are embedded in a vast amount of extracellular matrix having physical stiffness and elasticity, properties that support the mechanical load from bones and enable the flexible movement of synovial joints. Unlike chondrocytes that conduct the growth of long bones by forming the growth plate, articular chondrocytes show suppressed cell proliferation, unless these cells are exposed to pathological conditions such as mechanical overload. In the present study, we found that one of the members of the CCN family, CCN3, was significantly expressed in chondrocytes isolated from the epiphyseal head in developing rat synovial joints. Evaluation of the effect of recombinant CCN3 on those chondrocytes revealed that CCN3 promoted proteoglycan synthesis, whereas this factor repressed the proliferation of the same cells. These results suggest a critical role for CCN3 in the regulation of the biological properties of articular chondrocytes.

Polyhexanide and hydrogen peroxide inhibit proteoglycan synthesis of human chondrocytes

The use of local antiseptics is a common method in septic joint surgery. We tested polyhexanide and hydrogen peroxide, two of the most frequently used antiseptics with high efficacy and low toxicity. The purpose of this study was to evaluate the effects of both antiseptics on the extracellular cartilaginous matrix synthesis of human chondrocytes. Chondrocytes were isolated from donated human knee joints, embedded in alginate beads, and incubated for 10 and 30 minutes with polyhexanide (0·04%), hydrogen peroxide (3%), or phosphate-buffered saline (PBS) for control. Cartilaginous matrix production was quantified through light microscopic analysis of Alcian blue staining. Cell number and morphology were detected by histological analysis. Chondrocytes showed a decreased intensity of blue colouring after antiseptic treatment versus PBS. In contrast to that, neither the cell number per view field nor the cell morphology differed between the groups. Polyhexanide has more toxic potential than hydrogen peroxide. Based on the fact that the cell number and morphology was not altered by the substances at the examined concentrations, the lower intensity of Alcian blue staining of treated chondrocytes indicates a decreased cartilage-specific matrix synthesis by polyhexanide more than by hydrogen peroxide and control.

ウサギ脛骨骨端関節軟骨細胞の増殖およびプロテオグリカン産生に対するInterleukin-1βおよびnitric oxideの効果

ウサギ脛骨骨端関節軟骨細胞の増殖およびプロテオグリカン産生に対するInterleukin-1βおよびnitric oxideの効果

Effect of adiponectin on ATDC5 proliferation, differentiation and signaling pathways

Adiponectin, an adipose-secreted adipocytokine, exhibits various metabolic functions but has no known effect on bone development through the growth plate and specifically, in chondrocytes. Using the mouse ATDC5 cell line, a widely used in vitro model of chondrogenesis, we demonstrated the expression of adiponectin and its receptors during chondrogenic differentiation. Adiponectin at 0.5mug/ml increased chondrocyte proliferation, proteoglycan synthesis and matrix mineralization, as reflected by upregulation of the expression of type II collagen, aggrecan, Runx2 and type X collagen, and of alkaline phosphatase activity. Quantitative RT-PCR and gelatin zymography showed a significant increase in the matrix metalloproteinase MMP9's expression and activity following adiponectin treatment. We therefore concluded that adiponectin can directly stimulate chondrocyte proliferation and differentiation. To evaluate the underlying mechanisms, we examined the effect of adiponectin on the expression of chondrogenic signaling molecules: Ihh, PTHrP, Ptc1, FGF18, BMP7, IGF1 and p21 were all upregulated while FGF9 was downregulated. This study reveals novel and direct activity of adiponectin in chondrocytes, suggesting its positive effects on bone development.

Zonal Responsiveness of the Human Intervertebral Disc to Bone Morphogenetic Protein-2

In vitro experiment using bone morphogenetic protein-2 (BMP-2) and cells from the nucleus pulposus (NP), transitional zone (TZ), and anulus fibrosus (AF) of the human intervertebral disc (IVD). To demonstrate the differential effect of BMP-2 on DNA synthesis, proteoglycan synthesis, and osteocalcin mRNA expression in human IVD cells from the NP, TZ, and AF, respectively. BMP-2 has been proven to be effective in stimulating proteoglycan synthesis in articular chondrocytes and IVD cells from the NP. Nevertheless, the effect of BMP-2 on cells from different regions of the IVD has not yet been thoroughly elucidated. Human IVDs were harvested from surgical disc procedures and tissue from the NP, TZ, and AF was obtained. Disc tissue was enzymatically digested, and IVD cells were cultured three-dimensionally in alginate beads. Then IVD cell cultures from the NP, TZ, and AF were exposed to BMP-2. DNA synthesis and newly synthesized proteoglycan were measured. Reverse transcription-polymerase chain reaction for mRNA expression of bone sialoprotein, DLX5, osteocalcin, and collagen type I, was performed. Cells from the AF responded to BMP-2 with mitogenesis. There was no significant increase in DNA synthesis in cultures from the NP and TZ treated with BMP-2. Only cells from the NP showed a significant increase in newly synthesized proteoglycan in response to BMP-2. IVD cells from all zones demonstrated no significant expression of bone sialoprotein, DLX5, osteocalcin mRNA after treatment with BMP-2. BMP-2 clearly exerted a mitogenic effect on AF cells, and stimulated proteoglycan synthesis in NP cells. However, BMP-2 did not have an osteogenic effect in any IVD region. Taken together, these results confirm that BMP-2 can be used as an anabolic agent for mitogenesis in AF cells and NP cell matrix regeneration without the possibility of osteogenesis.

Cell–matrix interactions and dynamic mechanical loading influence chondrocyte gene expression and bioactivity in PEG-RGD hydrogels

The pericellular matrix (PCM) surrounding chondrocytes is thought to play an important role in transmitting biochemical and biomechanical signals to the cells, which regulates many cellular functions including tissue homeostasis. To better understand chondrocytes interactions with their PCM, three-dimensional poly(ethylene glycol) (PEG) hydrogels containing Arg–Gly–Asp (RGD), the cell-adhesion sequence found in fibronectin and which is present in the PCM of cartilage, were employed. RGD was incorporated into PEG hydrogels via tethers at 0.1, 0.4 and 0.8 mM concentrations. Bovine chondrocytes were encapsulated in the hydrogels and subjected to dynamic compressive strains (0.3 Hz, 18% amplitude strain) for 48 h, and their response assessed by cell morphology, ECM gene expression, cell proliferation and matrix synthesis. Incorporation of RGD did not influence cell morphology under free swelling conditions. However, the level of cell deformation upon an applied strain was greater in the presence of RGD. In the absence of dynamic loading, RGD appears to have a negative effect on chondrocyte phenotype, as seen by a 4.7-fold decrease in collagen II/collagen I expressions in 0.8 mM RGD constructs. However, RGD had little effect on early responses of chondrocytes (i.e. cell proliferation and matrix synthesis/deposition). When isolating RGD as a biomechanical cue, cellular response was very different. Chondrocyte phenotype (collagen II/collagen I ratio) and proteoglycan synthesis were enhanced with higher concentrations of RGD. Overall, our findings demonstrate that RGD ligands enhance cartilage-specific gene expression and matrix synthesis, but only when mechanically stimulated, suggesting that cell–matrix interactions mediate chondrocyte response to mechanical stimulation.

In VitroandIn VivoModulation of Cartilage Degradation by a StandardizedCentella asiaticaFraction

Osteoarthritis (OA) is a degenerative joint disease in which focal cartilage destruction is one of the primary features. The present study aims to evaluate the effect of a Centella asiatica fraction on in vitro and in vivo cartilage degradation. Bovine cartilage explants and bovine chondrocytes cultured in alginate were stimulated with IL-1 beta in the presence or absence of different concentrations (2, 5 and 10 microg/ml) of a standardized Centella asiatica triterpenes (CAT) fraction. The CAT fraction inhibited the IL-1 beta-induced proteoglycan (PG) release and nitric oxide (NO) production by cartilage explants in a dose-dependent manner. The IL-1 beta-induced reduction in PG synthesis and proliferation of chondrocytes cultured in alginate were counteracted by the CAT fraction at a concentration of 10 microg/ml. In a zymosan-induced acute arthritis model, the CAT fraction inhibited PG depletion without modulating joint swelling and inflammatory cell infiltration. In conclusion, the present study demonstrated for the first time that the tested Centella asiatica fraction was able to inhibit the zymosan-induced cartilage degradation in vivo without affecting the zymosan-induced inflammatory cell infiltration and joint swelling. The in vitro data indicate that the cartilage protective activity might at least partially be induced by the inhibition of NO production. The overall results indicate a possible disease modifying osteoarthritic activity of the Centella asiatica fraction.

Increased expression of the Akt/PKB inhibitor TRB3 in osteoarthritic chondrocytes inhibits insulin‐like growth factor 1–mediated cell survival and proteoglycan synthesis

The chondrocyte response to insulin-like growth factor 1 (IGF-1) is reduced with aging and in osteoarthritis (OA). IGF-1 signals through the phosphatidylinositol 3-kinase/Akt pathway. TRB3, a tribbles homolog, has been shown to inhibit IGF-1-mediated activation of Akt in HEK 293 cells. This study was undertaken to determine if TRB3 is expressed in chondrocytes, and whether the chondrocyte response to IGF-1 is reduced by TRB3. Human articular cartilage was obtained from normal tissue donors and from patients with OA at the time of knee replacement surgery. TRB3 was assessed in the tissue samples by reverse transcription-polymerase chain reaction, immunoblotting, and immunohistochemistry. Overexpression of TRB3 was induced by transient transfection to determine the effects of TRB3 on cell survival and proteoglycan synthesis. TRB3 messenger RNA was detected in normal human chondrocytes. TRB3 protein levels were low in cells from normal cartilage but significantly increased in cells from OA cartilage. Incubation with 2 agents that induce endoplasmic reticulum stress, tunicamycin and thapsigargin, increased TRB3 levels in normal cells. Overexpression of TRB3 inhibited Akt phosphorylation and reduced chondrocyte survival and proteoglycan synthesis. These results are the first to demonstrate that TRB3 is present in human chondrocytes, and that the level of TRB3 is increased in OA cartilage and in isolated OA chondrocytes. Because it is an inhibitor of Akt activation, elevated TRB3 production could play a role in the increased cell death and reduced response to IGF-1 observed in OA cartilage.

Mechanical compression of articular cartilage induces chondrocyte proliferation and inhibits proteoglycan synthesis by activation of the ERK pathway: implications for tissue engineering and regenerative medicine

Articular cartilage is recalcitrant to endogenous repair and regeneration and is thus a focus of tissue engineering and regenerative medicine strategies. A prerequisite for articular cartilage tissue engineering is an understanding of the signal transduction pathways involved in mechanical compression during trauma or disease. We sought to explore the role of the extracellular signal-regulated kinase 1/2 (ERK 1/2) pathway in chondrocyte proliferation and proteoglycan synthesis following acute mechanical compression. Bovine articular cartilage explants were cultured with and without the ERK 1/2 pathway inhibitor PD98059. Cartilage explants were statically loaded to 40% strain at a strain rate of 1/s for 5 s. Control explants were cultured under similar conditions but were not loaded. There were four experimental groups: (a) no load, without inhibitor; (b) no load, with the inhibitor PD98059; (c) loaded, without the inhibitor; and (d) loaded, with the inhibitor PD98059. The explants were cultured for varying durations from 5 min to 5 days and were then analysed by biochemical and immunohistochemical methods. Mechanical compression induced phosphorylation of ERK 1/2, and this was attenuated with the ERK 1/2 pathway inhibitor PD98059 in a dose-dependent manner. Chondrocyte proliferation was increased by mechanical compression. This effect was blocked by the inhibitor of the ERK 1/2 pathway. Mechanical compression also led to a decrease in proteoglycan synthesis that was reversed with inhibitor PD98059. In conclusion, the ERK 1/2 pathway is involved in the proliferative and biosynthetic response of chondrocytes following acute static mechanical compression.

CCN Family 2/Connective Tissue Growth Factor Modulates BMP Signalling as a Signal Conductor, Which Action Regulates the Proliferation and Differentiation of Chondrocytes

Both CCN family 2/connective tissue growth factor (CCN2/CTGF) and bone morphogenetic protein (BMP)-2 play an important role in cartilage metabolism. We evaluated whether or not CCN2 would interact with BMP-2, and examined the combination effect of CCN2 with BMP-2 (CCN2-BMP-2) on the proliferation and differentiation of chondrocytes. Immunoprecipitation-western blotting analysis, solid-phase binding assay and surface plasmon resonance (SPR) spectroscopy showed that CCN2 directly interacted with BMP-2 with a dissociation constant of 0.77 nM as evaluated by SPR. An in vivo study revealed that CCN2 was co-localized with BMP-2 at the pre-hypertrophic region in the E18.5 mouse growth plate. Interestingly, CCN2-BMP-2 did not affect the BMP-2/CCN2-induced phosphorylation of p38 MAPK but caused less phosphorylation of ERK1/2 in cultured chondrocytes. Consistent with these results, cell proliferation assay showed that CCN2-BMP-2 stimulated cell growth to a lesser degree than by either CCN2 or BMP-2 alone, whereas the expression of chondrocyte marker genes and proteoglycan synthesis, representing the mature chondrocytic phenotype, was increased collaboratively by CCN2-BMP-2 treatment in cultured chondrocytes. These findings suggest that CCN2 may regulate the proliferating and differentiation of chondrocytes by forming a complex with BMP-2 as a novel modulator of BMP signalling.

No improvement of joint cartilage healing after trauma by the administration of insulin-like growth factor I, epidermal growth factor and fibroblast growth factor in rabbits

Insulin-like growth factor I (Somatomedin C), epidermal growth factor, and fibroblast growth factor exert a stimulatory effect on articular chondrocyte metabolism in vitro. We studied the effect of these peptides on the repair of standardized full-thickness articular cartilage defects in the knee-joints of 32 young adult rabbits. In our in vivo study, the growth factors failed to stimulate cartilage cell replication, chondrocyte proteoglycan synthesis or defect filling. The trauma, however, had a significant stimulatory effect on the mitotic activity of the chondrocytes near the defect, but not on the synthesis of sulfated proteoglycans.

Mechanical regulation of proteoglycan synthesis in normal and osteoarthritic human articular chondrocytes – roles for α5 and αVβ5 integrins

The importance of biomechanical forces in regulating normal chondrocyte metabolism is well established and the mechanisms whereby mechanical forces are transduced into biochemical responses by chondrocytes are beginning to be understood. Previous studies have indicated that cyclical mechanical stimulation induces increased aggrecan gene expression in normal but not osteoarthritic chondrocytes in monolayer. It remains unclear, however, whether these effects on gene expression are associated with changes in proteoglycan production and whether any changes in proteoglycan expression is dependent on integrins or integrin associated proteins. Normal and osteoarthritic articular chondrocytes in monolayer were exposed to 0.33 Hz mechanical stimulation for 20 min in the absence or presence of function modifying anti-integrin antibodies. Following stimulation GAG and proteoglycan (PG) synthesis was assessed by DMMB assay and western blotting. Mechanical stimulation of normal chondrocytes resulted in increased GAG synthesis that was blocked by the presence of antibodies to alpha5 and alphaVbeta5 integrins and CD47. Electrophoretic patterns of PGs released from normal chondrocytes following mechanical stimulation showed an increase in newly-synthesized aggrecan that was not fragmented or degraded. Chondrocytes from osteoarthritic cartilage showed lower levels of GAG production when compared to normal chondrocytes and synthesis was not influenced by mechanical stimulation. These studies show that chondrocytes derived from normal and OA cartilage have different matrix production responses to mechanical stimulation and suggest previously unrecognised roles for alphaVbeta5 integrin in regulation of chondrocyte responses to biomechanical stimulation.

Modulation of articular chondrocyte proliferation and anionic glycoconjugate synthesis by glucosamine (GlcN), N-acetyl GlcN (GlcNAc) GlcN sulfate salt (GlcN.S) and covalent glucosamine sulfates (GlcN-SO 4)

To investigate, in chondrocyte cultures under conditions for maximizing responses in proliferation and proteoglycan (PG) synthesis, the effects of glucosamine hydrochloride (GlcN.HCl) and glucosamine sulfate (GlcN.S) salts, N-acetyl glucosamine (GlcNAc), and covalently substituted GlcN-X,Y,Z(SO(4))(n) (general formula). Bovine articular chondrocytes (BAC) were studied under anchorage-independent (AI, alginate beads) and anchorage-dependent (AD, plastic surface) conditions. Differentiation markers were evaluated (e.g., cartilage-specific (V+C)(-) fibronectin). Varying concentrations of GlcN.HCl, GlcN.S, GlcNAc and GlcN sulfated at positions -2, -3, -6, (-2,3), (-3,6) and (-3,4,6), were tested. Cell proliferation, DNA synthesis and [(35)S]-sulfate incorporation into newly synthesized PG were determined. Increasing GlcN.HCl or GlcN.S concentrations gave decreasing net PG synthesis. Compounds showed more pronounced effects in AD cultures (expressing the V(-)C(+) fibronectin isoform) compared to AI cultures ((V+C)(-) isoform). Addition of GlcN.HCl or GlcN.S gave a concentration-dependent decrease in BAC proliferation, partially prevented by glucose (Glc). GlcNAc was not inhibitory. Addition of GlcN-2-SO(4) or GlcN-2,6-diSO(4) did not affect proliferation or DNA synthesis. The other GlcN-sulfates gave varying inhibitory effects, which for GlcN-3-SO(4) were reversed by inosine. The free amino group of GlcN seems responsible for inhibition of chondrocyte proliferation and PG synthesis. These effects were greater under higher concentrations of GlcN in AD vs AI conditions. GlcN.HCl behaves similarly to GlcN.S, but differential effects with GlcN-X,Y,Z(SO(4))(n) isomers were observed. Acetylation or sulfation of the GlcN amino group reverses or partially reverses, respectively, anti-proliferative effects of GlcN. Sulfation of GlcN, at positions 3 and 6 results in complex effects on AC proliferation and PG synthesis.