Aggrecan Synthesis Research Articles

234 OPTIMIZED ALKYLATED CYCLODEXTRIN POLYSULPHATES RESTORE OSTEOARTHRITIC CHONDROCYTE EXTRACELLULAR MATRIX METABOLISM

Objectives: To compare the ability of different cyclodextrin polysulphate derivatives to affect human articular cartilage cell metabolism in vitro and to act as Disease Modifying OsteoArthritis Drugs in vivo. Methods: OA chondrocytes were cultured in gelled alginate and exposed to 5 µg/ml of 2,3,6-tri-O-methyl-β-cyclodextrin (ME-CD), 2,3-di-O-methyl-6-sulphate-β-cyclodextrin (ME-CD-6-S), 2,6-di-O-methyl-3-sulphate-β-cyclodextrin (ME-CD-3-S), (2-carboxyethyl)-β-cyclodextrin polysulphate (CE-CDPS), (2-hydroxypropyl)-β-cyclodextrin polysulphate (HP-CDPS), 6-monoamino-6-monodeoxy-β-cyclodextrin polysulphate (MA-CDPS) or β-cyclodextrin polysulphate (CDPS) during 5 days. Effects on IL-1-driven chondrocyte extracellular matrix (ECM) metabolism was assayed by analysis of the accumulation of aggrecan in the interterritorial matrix and by the release of IL-6 in the culture supernatant. MA-CDPS, HP-CDPS, CE-CDPS and CDPS were analyzed for their in vitro effect on coagulation and their ability to activate platelets in an in vitro assay to detect possible crossreactivity with HIT (heparin-induced thrombocytopenia) antibodies. CE-CDPS was selected for further qPCR analyses, in order to investigate whether this compound directly influences the gene expression of IL-6 and aggrecan. Results: The monosulphated cyclodextrins ME-CD-6-S and -3S failed to affect aggrecan synthesis and IL-6 secretion by the OA chondrocytes. Polysulphated cyclodextrins MA-CDPS, HP-CDPS, CE-CDPS and CDPS at 5 µg/ml concentrations, on the other hand, significantly induced aggrecan production and repressed IL-6 release by the chondrocytes in culture. Five µg/ml of CE-CDPS, in contrast to MA-CDPS, HP-CDPS and CDPS, did not significantly activate platelets and thus showed no potential to induce HIT thromboembolic accidents in vivo. Therefore, CE-CDPS was selected for further qPCR analyses. These analyses confirmed the anabolic and anti-catabolic profile of CE-CDPS. Conclusions: CE-CDPS is a new, structurally adjusted sulphated β-cyclodextrin derivative with preserved chondroprotective capacity and a promising safety profile.

Serum biomarker levels for musculoskeletal disease in two‐ and three‐year‐old racing Thoroughbred horses: A prospective study of 130 horses

Biomarkers have shown some in vivo promise for the detection of musculoskeletal injuries, but further study to assess biomarker levels in clinical orthopaedic disease is required. To assess 7 serum biomarkers for the detection of musculoskeletal injuries. Two- and 3-year-old racehorses were entered into the study (n = 238). Exit criteria were lack of training for >30 days, or completion of 10 study months. Data from horses with solitary musculoskeletal injuries and completion of >2 months were analysed. Musculoskeletal injury was considered intra-articular fragmentation (IAF), tendon or ligamentous injury (TL), stress fractures (SF) and dorsal metacarpal disease (DMD). Monthly lameness examination and serum collection were performed. Serum was analysed for glycosaminoglycan (GAG), type I and II collagen degradation (C1, 2C), type II collagen synthesis (CPII), type II collagen degradation (Col CEQ), aggrecan synthesis (CS846), osteocalcin (OC) as a marker of bone formation and (C-terminal telopeptide of type I collagen) CTX as a marker of bone degradation. Of the 238 horses 59 injured and 71 uninjured control horses met the analysis criteria. Based on injury no significant differences in the proportions were observed for age, gender or lesion type, although a higher proportion of injuries occurred at the beginning of the study. Of injured horses, 16 (27%) sustained an IAF, 17 (29%) a TL injury, 7 (12%) SF and 19 (32%) were diagnosed with DMD. There were significant changes seen in biomarkers based on the injury incurred when longitudinal samples were assessed. Furthermore, based on the serum biomarkers collected prior to injury, horses could be correctly classified as injured or uninjured 73.8% of the time. A unique biomarker pattern occurred before each injury and this was beneficial in classifying horses as injured or uninjured. Biomarkers have the potential to be used as a screening aid prior to musculoskeletal injury.

Enhancement of intervertebral disc cell senescence by WNT/β‐catenin signaling–induced matrix metalloproteinase expression

To determine whether intervertebral disc (IVD) cells express β-catenin and to assess the role of the WNT/β-catenin signaling pathway in cellular senescence and aggrecan synthesis. The expression of β-catenin messenger RNA (mRNA) and protein in rat IVD cells was assessed by using several real-time reverse transcription-polymerase chain reaction, Western blot, immunohistochemical, and immunofluorescence analyses. The effect of WNT/β-catenin on nucleus pulposus (NP) cells was examined by transfection experiments, an MTT assay, senescence-associated β-galactosidase staining, a cell cycle analysis, and a transforming growth factor (TGFβ)/bone morphogenetic protein (BMP) pathway-focused microarray analysis. We found that β-catenin mRNA and protein were expressed in discs in vivo and that rat NP cells exhibited increased β-catenin mRNA and protein upon stimulation with lithium chloride, a known activator of WNT signaling. LiCl treatment inhibited the proliferation of NP cells in a time- and dose-dependent manner. In addition, there was an increased level of cellular senescence in LiCl-treated cells. Long-term treatment with LiCl induced cell cycle arrest and promoted subsequent apoptosis in NP cells. Activation of WNT/β-catenin signaling also regulated the expression of aggrecan. We also demonstrated that WNT/β-catenin signaling induced the expression of matrix metalloproteinases (MMPs) and TGFβ in NP cells. The activation of WNT/β-catenin signaling promotes cellular senescence and may modulate MMP and TGFβ signaling in NP cells. We hypothesize that the activation of WNT/β-catenin signaling may lead to an increased breakdown of the matrix, thereby promoting IVD degeneration.

Change in proteoglycan metabolism is a characteristic of human patellar tendinopathy

To determine differences in the metabolism of proteoglycans and the gene expression of proteinases and their inhibitors between patellar tendons exhibiting chronic overuse tendinopathy and normal patellar tendons in humans. Rates of loss and synthesis of proteoglycans were determined. Radiolabeled and total proteoglycans retained in and lost from the tissue were analyzed by fluorography and Western blotting. Levels of messenger RNA for matrix metalloproteinase 1 (MMP-1), MMP-2, MMP-3, MMP-9, MMP-13, ADAMTS-1, ADAMTS-4, ADAMTS-5, tissue inhibitor of metalloproteinases 1 (TIMP-1), TIMP-2, TIMP-3, and TIMP-4 were determined in fresh tissue. The rate of loss of (35)S-labeled proteoglycans was greater in abnormal tendons, as was the rate of synthesis of proteoglycans. Fluorography and Western blotting revealed the presence of greater amounts of large proteoglycans (aggrecan and versican) in abnormal tendons, and these proteoglycans were rapidly lost from the matrix of abnormal tendons. There was no significant difference in the expression of ADAMTS-1, ADAMTS-4, ADAMTS-5, MMP-1, MMP-2, MMP-3, MMP-13, TIMP-2, TIMP-3, or TIMP-4. There was a significant increase in the expression of MMP-9 and TIMP-1 in abnormal tendons. Our findings suggest that a change in the proteoglycan content of the extracellular matrix in abnormal tendons results from the altered metabolism of the cells, reflected in the enhanced synthesis of the large proteoglycans aggrecan and versican, and does not appear to result from changes at the level of gene expression.

BMP-2 and TGF-β stimulate expression of β1,3-glucuronosyl transferase 1 (GlcAT-1) in nucleus pulposus cells through AP1, TonEBP, and Sp1: Role of MAPKs

The goal of the study was to investigate bone morphogenetic protein 2 (BMP-2) and transforming growth factor β (TGF-β) control of the expression of β1,3-glucuronosyl transferase 1 (GlcAT-1), an important regulator of chondroitin sulfate synthesis in cells of the nucleus pulposus. Treatment with both growth factors resulted in induction of GlcAT-1 expression and promoter activity. Deletion analysis indicated that promoter constructs lacking AP1 and TonE sites were unresponsive to growth factor treatment. Experiments using dominant-negative proteins showed that these transcription factors along with Sp1 were required for induction of GlcAT-1 promoter activity. Moreover, when either AP1 or TonE binding sites were mutated, induction was suppressed. Both BMP-2 and TGF-β increased c-Jun and TonEBP expression and phosphorylation of transactivation domains. We investigated the role of the mitogen-activated protein kinase (MAPK) signaling pathway following growth factor treatment; a robust and transient activation of ERK1/2, p38, and JNK was noted. Treatment with MAPK inhibitors blocked BMP-2- and TGF-β-induced AP1 reporter function, GlcAT-1 expression, and GAG accumulation. We found that DN-ERK1 but not DN-ERK2 resulted in suppression of growth factor–mediated induction of GlcAT-1 promoter activity; we also showed that p38δ was important in GlcAT-1 activation. Results of these studies demonstrate that BMP-2 and TGF-β regulate GlcAT-1 expression in nucleus pulposus cells through a signaling network comprising MAPK, AP1, Sp1, and TonEBP. It is concluded that by controlling both GAG and aggrecan synthesis, these growth factors positively influence disk cell function. © 2010 American Society for Bone and Mineral Research.

Inhibitory effect of lactoferrin on hypertrophic differentiation of ATDC5 mouse chondroprogenitor cells

The skeleton is formed by two different mechanisms. In intramembranous ossification, osteoblasts form bone directly, whereas in endochondral ossification, chondrocytes develop a cartilage template, prior to osteoblast-mediated skeletogenesis. Lactoferrin is an iron-binding glycoprotein belonging to the transferrin family. It is known to promote the growth and differentiation of osteoblasts. In this study, we investigated the effects of bovine lactoferrin on the chondrogenic differentiation of ATDC5 chondroprogenitor cells. This mouse embryonic carcinoma-derived clonal cell line provides an in vitro model of chondrogenesis. Lactoferrin treatment of differentiating ATDC5 cells promoted cell proliferation in the initial stage of the differentiation process. However, lactoferrin treatment resulted in inhibition of hypertrophic differentiation, characterized by suppression of alkaline phosphatase activity, aggrecan synthesis and N-cadherin expression. This inhibitory effect was accompanied by sustained Sox9 expression, as well as increased Smad2/3 expression and phosphorylation, suggesting that lactoferrin regulates chondrogenic differentiation by up-regulating the Smad2/3-Sox9 signaling pathway.

Chondrocytes and Meniscal Fibrochondrocytes Differentially Process Aggrecan During De Novo Extracellular Matrix Assembly

Aggrecan is an extracellular matrix molecule that contributes to the mechanical properties of articular cartilage and meniscal fibrocartilage, but the abundance and processing of aggrecan in these tissues are different. The objective of this study was to compare patterns of aggrecan processing by chondrocytes and meniscal fibrochondrocytes in tissue explants and cell-agarose constructs. The effects of transforming growth factor-beta 1 (TGF-beta1) stimulation on aggrecan deposition and processing were examined, and construct mechanical properties were measured. Fibrochondrocytes synthesized and retained less proteoglycans than did chondrocytes in tissue explants and agarose constructs. In chondrocyte constructs, TGF-beta1 induced the accumulation of a 120-kDa aggrecan species previously detected in mature bovine cartilage. Fibrochondrocyte-seeded constructs contained high-molecular-weight aggrecan but lacked aggrecanase-generated fragments found in native, immature meniscus. In addition, reflecting the lesser matrix accumulation, fibrochondrocyte constructs had significantly lower compression moduli than did chondrocyte constructs. These cell type-specific differences in aggrecan synthesis, retention, and processing may have implications for the development of functional engineered tissue grafts.

Activation of TonEBP by Calcium Controls β1,3-Glucuronosyltransferase-I Expression, a Key Regulator of Glycosaminoglycan Synthesis in Cells of the Intervertebral Disc

The goal of this investigation was to study the expression and regulation of beta1,3-Glucuronosyltransferase-I (GlcAT-I), a key enzyme regulating GAG synthesis in cells of the intervertebral disc. There was a robust expression of GlcAT-I in the nucleus pulposus in vivo. Treatment with the calcium ionophore ionomycin resulted in increased GlcAT-I expression, whereas GlcAT-I promoter constructs lacking TonE site or a mutant TonE were unresponsive to the ionophore. Experiments using TonEBP and DN-TonEBP constructs showed that TonEBP positively regulated GlcAT-I promoter activity. ChIP analysis confirmed binding of TonEBP to the promoter. We further validated the role of TonEBP in controlling GlcAT-I expression using mouse embryo fibroblasts from TonEBP null mice. GlcAT-I promoter activity in null cells was significantly lower than the wild type cells. In contrast to wild type cells, treatment with ionomycin failed to increase GlcAT-I promoter activity in null cells. We then investigated if calcineurin (Cn)-NFAT signaling played a regulatory role in GlcAT-I expression. Inhibition of Cn following ionomycin treatment did not block GlcAT-I and tauT, a TonEBP-responsive reporter activity. GlcAT-I promoter activity was suppressed by co-expression of Cn, NFAT2, NFAT3, and NFAT4. Moreover, following ionomycin treatment, fibroblasts from CnAalpha and CnAbeta null mice exhibited robust induction in GlcAT-I promoter activity compared with wild type cells. Results of these studies demonstrate that calcium regulates GlcAT-I expression in cells of the nucleus pulposus through a signaling network comprising both activator and suppressor molecules. The results suggest that by controlling both GAG and aggrecan synthesis, disc cells can autoregulate their osmotic environment and accommodate mechanical loading.

IGF-1 does not moderate the time-dependent transcriptional patterns of key homeostatic genes induced by sustained compression of bovine cartilage

To determine changes in chondrocyte transcription of a range of anabolic, catabolic and signaling genes following simultaneous treatment of cartilage with Insulin-like growth factor-1 (IGF-1) and ramp-and-hold mechanical compression, and compare with effects on biosynthesis. Explant disks of bovine calf cartilage were slowly compressed (unconfined) over 3-min to their 1mm cut-thickness (0%-compression) or to 50%-compression with or without 300 ng/ml IGF-1. Expression of 24 genes involved in cartilage homeostasis was measured using qPCR at 2, 8, 24, 32, 48 h after compression +/-IGF-1. Clustering analysis was used to identify groups of co-expressed genes to further elucidate mechanistic pathways. IGF-1 alone stimulated gene expression of aggrecan and collagen II, but simultaneous 24h compression suppressed this effect. Compression alone up-regulated expression of matrix metalloproteinase (MMP)-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motif (ADAMTS)-5 and transforming growth factor (TGF)-beta, an effect not reversed by simultaneous IGF-1 treatment. Temporal changes in expression following IGF-1 treatment were generally slower than that following compression. Clustering analysis revealed five distinct groups within which the pairings, tissue inhibitor of metalloproteinase (TIMP)-3 and ADAMTS-5, MMP-1 and IGF-2, and IGF-1 and Collagen II, were all robustly co-expressed, suggesting inherent regulation and feedback in chondrocyte gene expression. While aggrecan synthesis was transcriptionally regulated by IGF-1, inhibition of aggrecan synthesis by sustained compression appeared post-transcriptionally regulated. Sustained compression markedly altered the effects of IGF-1 on expression of genes involved in cartilage homeostasis, while IGF-1 was largely unable to moderate the transcriptional effects of compression alone. The demonstrated co-expressed gene pairings suggest a balance of anabolic and catabolic activity following simultaneous mechanical and growth factor stimuli.

Effects of Pulsed Low-Intensity Ultrasound on Human Child Chondrocytes

The effect of pulsed low-intensity ultrasound (PLIUS) on human articular chondrocytes was evaluated in an in vitro 3-D agarose gel culture model. Chondrocytes isolated from young children's articular cartilage of ablated polydactylia were embedded in gel after expansion and exposed to PLIUS on the third day after embedding. Another group of cells was exposed to sham PLIUS as a control. Different intensities of PLIUS treatment—18 mW/cm 2, 48 mW/cm 2, 72 mW/cm 2 and 98 mW/cm 2 (1.0 MHz, with burst duration of 200 μs repeated at 1.0 kHz)—were administered for 20 min/d, and the medium was replaced twice a week. The cultures were evaluated for aggrecan synthesis by enzyme-linked immunosorbent assay (ELISA), type II collagen production by Western blotting or ELISA and cell proliferation by total DNA measurement. The PLIUS was found to increase aggrecan synthesis in a time-dependent manner. The maximal response was observed at an intensity of 48 mW/cm 2. After 14 d of exposure at this intensity, the aggrecan synthesis was 214 ± 26% of control, and type II collagen synthesis was 148.5 ± 8.0% of control. However, PLIUS treatment revealed no significant influence on cell proliferation, confirming that the stimulation of aggrecan and type II collagen synthesis by PLIUS was not the result of an increase in chondrocyte cell proliferation. In addition, it was found that human chondrocytes harvested from older donors become less responsive to PLIUS. From this in vitro 3-D study of cultured human chondrocytes, our findings suggest that PLIUS may be applied to the tissue engineering of cartilage constructs. (E-mail: d740113@kmu.edu.tw)

High levels of serum IL-18 promote cartilage loss through suppression of aggrecan synthesis

Osteoarthritis (OA) is closely related to the function of several inflammatory cytokines. It has been reported that older age is associated with higher serum levels of the inflammatory cytokine IL-18. In the present study, we investigated the long-term role of serum IL-18 in cartilage loss in vivo using a new strain of IL-18 transgenic mouse (Tg) in comparison with wild-type (WT) mice. The IL-18 Tg mouse strain we developed constitutively overproduces soluble mature IL-18 in the lungs but not in other tissues, including joints. These Tg mice showed high levels of serum IL-18, but not IL-1β. No inflammatory cells, fibrillation or synovitis were observed in the knee joints of either IL-18 Tg or WT mice. However, the cartilage cellularity of the femoral and tibial condyles of IL-18 Tg mice was significantly reduced in comparison with control WT mice. Aggrecan was detected in only a few cells in the deep zone of the articular cartilage of Tg mice. The expression of aggrecan mRNA was also significantly decreased in articular chondrocytes from Tg mice when compared with WT mice. In contrast, endogenous IL-18 mRNA was significantly increased in the chondrocytes of Tg mice in comparison with WT mice. Expression of IFN-γ was also significantly increased in the Tg mice. Moreover, IL-18 transgene-positive caspase-1-deficient mice showed articular cartilage loss that was independent of endogenous IL-1β. In cultured chondrocytes isolated from WT mice, the expression of aggrecan mRNA was dosage-dependently suppressed by treatment with recombinant IL-18. In contrast, IL-18 stimulated the expression of mRNA for endogenous IL-18 and IFN-γ. These results suggest that high levels of serum IL-18 promote the overexpression of endogenous IL-18 in articular chondrocytes, resulting in cartilage loss through suppression of aggrecan synthesis. Thus IL-18 may play an important role in the pathogenesis of articular cartilage loss in osteoarthritis.

The Carbon Monoxide-Releasing Molecule Tricarbonyldichlororuthenium(II) Dimer Protects Human Osteoarthritic Chondrocytes and Cartilage from the Catabolic Actions of Interleukin-1β

We have investigated the effects of a carbon monoxide-releasing molecule, tricarbonyldichlororuthenium(II) dimer (CORM-2), on catabolic processes in human osteoarthritis (OA) cartilage and chondrocytes activated with interleukin-1beta. In these cells, proinflammatory cytokines induce the synthesis of matrix metalloproteinases (MMPs) and aggrecanases, including members of a disintegrin and metalloproteinase with thrombospondin domain (ADAMTS) family, which may contribute to cartilage loss. CORM-2 down-regulated MMP-1, MMP-3, MMP-10, MMP-13, and ADAMTS-5 in OA chondrocytes, and it inhibited cartilage degradation. These effects were accompanied by increased aggrecan synthesis and collagen II expression in chondrocytes. Our results also indicate that the inhibition of extracellular signal-regulated kinase 1/2 and p38 activation by CORM-2 may contribute to the maintenance of extracellular matrix homeostasis. These observations suggest that CORM-2 could exert chondroprotective effects due to the inhibition of catabolic activities and the enhancement of aggrecan synthesis.

In vivo intervertebral disc remodeling: Kinetics of mRNA expression in response to a single loading event

Kinetics of mRNA expression following a single loading event was measured using an in vivo rat tail model. Animals were instrumented and loaded in compression for 1.5 h at 1 MPa and 1 Hz. Real-time RT-PCR was used to measure mRNA levels 0, 8, 24 and 72 h after mechanical stimulation for genes associated with matrix proteins (aggrecan, collagen-I, collagen-II), proteases (MMP-2, MMP-3, MMP-13, ADAMTS-4), and their inhibitors (TIMP-1, TIMP-3) in anulus fibrosus and nucleus pulposus regions. Baseline mRNA levels were of greatest abundance for matrix proteins and lowest for proteases. The mRNA levels reached maximum levels 24 h following mechanical stimulation for the majority of genes evaluated, but some had maximum levels 8 and 72 h following loading. The mRNA levels returned to baseline levels for all genes in the nucleus 72 h following loading, but the majority of genes in the anulus remained upregulated. Results support a coordinated strategy of relative mRNA expression that varied over time beginning with inhibition of tissue breakdown, followed by synthesis of aggrecan and matrix degrading enzymes, and eventually collagen metabolism days following loading. Consequently, optimal time for tissue harvest for mRNA measurements depends on genes of interest. Results suggest attempts at anabolic remodeling must be given adequate time for metabolic processes and protein synthesis to occur, and that changes in TIMP and MMP levels may have greater potency in affecting structural protein abundance than direct changes in the structural protein messages. Results have important implications for disc remodeling and tissue engineering.

Promotion of the articular cartilage proteoglycan degradation by T-2 toxin and selenium protective effect

To identify the relationship between T-2 toxin and Kashin-Beck disease (KBD), the effects of T-2 toxin on aggrecan metabolism in human chondrocytes and cartilage were investigated in vitro. Chondrocytes were isolated from human articular cartilage and cultured in vitro. Hyaluronic acid (HA), soluble CD44 (sCD44), IL-1beta and TNF-alpha levels in supernatants were measured by enzyme-linked immunosorbent assay (ELISA). CD44 content in chondrocyte membrane was determined by flow cytometry (FCM). CD44, hyaluronic acid synthetase-2 (HAS-2) and aggrecanases mRNA levels in chondrocytes were determined using reverse transcription polymerase chain reaction (RT-PCR). Immunocytochemical method was used to investigate expressions of BC-13, 3-B-3(-) and 2-B-6 epitopes in the cartilage reconstructed in vitro. T-2 toxin inhibited CD44, HAS-2, and aggrecan mRNA expressions, but promoted aggrecanase-2 mRNA expression. Meanwhile, CD44 expression was found to be the lowest in the chondrocytes cultured with T-2 toxin and the highest in control plus selenium group. In addition, ELISA results indicated that there were higher sCD44, IL-1beta and TNF-alpha levels in T-2 toxin group. Similarly, higher HA levels were also observed in T-2 toxin group using radioimmunoprecipitation assay (RIPA). Furthermore, using monoclonal antibodies BC-13, 3-B-3 and 2-B-6, strong positive immunostaining was found in the reconstructed cartilage cultured with T-2 toxin, whereas no positive staining or very weak staining was observed in the cartilage cultured without T-2 toxin. Selenium could partly inhibit the effects of T-2 toxin above. T-2 toxin could inhibit aggrecan synthesis, promote aggrecanases and pro-inflammatory cytokines production, and consequently induce aggrecan degradation in chondrocytes. These will perturb metabolism balance between aggrecan synthesis and degradation in cartilage, inducing aggrecan loss in the end, which may be the initiation of the cartilage degradation.

Inhibition of hyaluronan export reduces collagen degradation in interleukin-1 treated cartilage

BackgroundOsteoarthrosis is characterized by cartilage erosion, proteolysis of aggrecan and collagen, and disturbed rates of synthesis of aggrecan and hyaluronan by chondrocytes, with hyaluronan over-production being an early reaction. We considered that inhibition of hyaluronan export might prevent subsequent proteoglycan loss and collagen degradation.MethodsTo test this hypothesis, we studied a tissue culture model using bovine cartilages explants activated with IL-1α to induce osteoarthritic reactions using the phosphodiesterase-5 inhibitors tadalafil, zaprinast and vardenafil.ResultsThese drugs inhibited hyaluronan export, but they did not inhibit hyaluronan synthase activity. Simultaneously, they inhibited proteoglycan loss and collagen degradation, but not their synthesis. They also reduced the release of gelatinases into the culture media and diffusion of the indicator protein horseradish peroxidase through the cartilage explants. The mechanism of action of these compounds may be through inhibition of hyaluronan exporter multidrug resistance-associated protein 5 (MRP5), because the effective drug concentrations were much higher than required for phosphodiesterase-5 inhibition and intracellular cGMP accumulation.ConclusionInhibition of hyaluronan over-production may be an appropriate target to attenuate IL-1-induced reactions in osteoarthritic cartilage.

Effect of cell density on the rate of glycosaminoglycan accumulation by disc and cartilage cells in vitro

Aggrecan concentration falls markedly during cartilage and disc degeneration with unfortunate biomechanical and physiological consequences. There is thus now an increasing interest in developing biological methods for its replacement. One approach is to stimulate aggrecan and hence glycosaminoglycan (GAG) production by resident cells through growth factor or genetic engineering. Another approach is to implant autologous cells into the cartilage or disc to enhance GAG production. In both instances GAG accumulation depends both on the number of active cells in the cartilage or disc and the rate of aggrecan synthesis per cell. Here we examine how cell density influences the rate of glycosaminoglycan accumulation in a three-dimensional cell culture system. In the results, at cell densities found in vivo (standard condition) in the articular cartilage and the disc nucleus viz. 4 million cells/ml and at 21% oxygen the concentration of GAG in the bead reached 520.9+/-62.4 microg/ml and 649.0+/-40.1 microg/ml, respectively, in 5 days. These concentrations could be increased to 2-4-fold by raising cell density to 25 million cells/ml. However, rates of GAG production per cell decreased by 50-60%. These results showed that rate of accumulation of glycosaminoglycan in this culture system in vitro is slow and is limited both by the rate of production of GAG per cell and by the cell density which can be maintained in a viable state. Although GAG production can be increased somewhat by use of higher cell densities, the consequent fall in concentration of oxygen and other nutrients in the center of three-dimensional constructs slows metabolism and leads to apoptosis and cell death, which again limits the rate that the cells can produce matrix.

Mechanical Chondroplasty: Early Metabolic Consequences In Vitro

The purpose of this study was to determine the depth of penetration from mechanical chondroplasty and metabolic consequences of this procedure on the remaining articular cartilage. Mechanical chondroplasty was performed in vitro on a portion of fresh grade I or II articular cartilage from 8 human knee arthroplasty specimens. Treated and control (untreated) explants (approximately 30 mg) were cut from the cartilage. The explants were divided into 2 groups, day 1 and day 4, placed separately in a 48-well plate containing media, and incubated at 37 degrees C for 24 hours. After the 24-hour incubation, the explants were weighed on day 1 and day 4, and explant media were removed and tested for total proteoglycan synthesis and aggrecan synthesis. At time 0, 2 sets (2.6 mm each) of treated and control cartilage slices were cut with a precision saw. One set was stained for confocal laser microscopy via a cytotoxicity stain to determine cell viability. The second set was stained with H&E to determine depth of penetration. The mean depth of penetration was 252.8 +/- 78 microm. There was no significant difference (P > .25) between total proteoglycan synthesis for control versus treatment groups on day 1 or 4. Aggrecan synthesis was significantly reduced on day 1 when normalized for tissue weight (P = .019) and double-stranded deoxyribonucleic acid (P = .004). On day 4, no significant difference was detected. Confocal laser microscopy did not show cell death below the zone of treatment. There was no significant metabolic consequence caused by chondroplasty to the remaining articular cartilage, and the zone of injury was limited to the treatment area. Mechanical chondroplasty causes no significant metabolic consequences to articular cartilage under these conditions.

Chondrogenic Differentiation of Human Articular Chondrocytes Differs in Biodegradable PGA/PLA Scaffolds

Cartilage tissue engineering is applied clinically to cover and regenerate articular cartilage defects. Two bioresorbable nonwoven scaffolds, polyglycolic acid (PGA) and poly(lactic-co-glycolic acid) (PLGA) (90/10 copolymer of L-lactide and glycolide), were seeded with human chondrocytes after initial progeny in a monolayer with a serum-free medium. Two subgroups of nontreated and plasma-treated (using low-pressure plasma technique) scaffolds were investigated. The constructs were cultivated after seeding in six-well plates with serum-free medium for 7 days and implanted subcutaneously into nude mice for 6 and 12 weeks. Chondrogenic differentiations were investigated using immunhistology and reverse transcriptase-polymerase chain reaction. Cell adhesion only differed from 50% to 65% without a significant difference between the groups. During further cultivation for 7 days, the aggrecan synthesis of the seeded constructs was always higher in the PGA groups (p < 0.05). The mRNA gene expression for collagen type II was significantly higher in the PGA groups after 6 and 12 weeks (p < 0.05). A decrease in the expression of collagen type I was investigated in all groups. The expression for collagen type X and cartilage oligomeric matrix protein (COMP) increased in all groups over time. After cell proliferation in serum-free medium, the long-term chondrogenic differentiation in PGA scaffolds in vitro is cartilage specific and may be utilized in cartilage tissue engineering applications.

Biosynthesis of glycosaminoglycans and aggrecan by tumor cells in salivary pleomorphic adenoma: ultrastructural evidence.

The present study attempted to discover the sites of synthesis of various glycosaminoglycans (GAGs) and aggrecan in salivary pleomorphic adenoma (PA) with the use of a highly sensitive and specific post-embedding immunogold-silver staining technique at the ultrastructural level. Silver particles representing various GAGs and aggrecan were found to accumulate frequently in the intercellular spaces of non-luminal cells in the epithelial clusters and were dispersed in the myxoid matrix of the mesenchyme-like areas. Furthermore, the non-luminal epithelial cells were demonstrated to contain immunopositive intracytoplasmic vesicles and vacuoles, some of which were of Golgi complex origin. In contrast, intracellular silver particles for hyaluronic acid were mostly attached to the inner surface of the cell membrane. These observations agree well with the current theories of the biosynthesis of GAGs and proteoglycans and provide direct evidence for the production of various GAGs and aggrecan by tumor epithelial cells of PA. Such findings support the ideas that in PA a loss of epithelium occurs by stromalization following epithelial secretion of extracellular matrix substances and transformation of epithelium to mesenchyme represents the basic principle of the tissue heterogeneity in this tumor.

Chondroitin Sulfate N-Acetylgalactosaminyltransferase-1 Plays a Critical Role in Chondroitin Sulfate Synthesis in Cartilage

Cartilage destruction leads to severe joint diseases, such as osteoarthritis and spinal disorders with back pain, and cartilage regeneration is very inefficient. A major component of the cartilage extracellular matrix is the proteoglycan aggrecan that contains approximately 100 chondroitin sulfate (CS) chains, which impart water absorption and resistance to compression. Here, we demonstrate that chondroitin sulfate N-acetylgalactosaminyltransferase-1 (CSGalNAcT-1) plays a critical role in CS biosynthesis in cartilage. By in situ hybridization and real time reverse transcription-PCR of developing cartilage, CSGalNAcT-1 exhibited the highest level of expression. Its expression in chondrogenic ATDC5 cells correlated well with that of aggrecan core protein. In heterozygote and homozygote aggrecan-null cartilage where aggrecan transcription is decreased, CSGalNAcT-1 transcription diminished accordingly. Overexpression of the enzyme in chondrocytic cells further enhanced CS biosynthesis but not that of the aggrecan core protein, indicating that the enzyme activity is not saturated in the cells and that aggrecan synthesized in the overexpressing cells is heavier than the native molecule. Analysis of the CS chains synthesized in the overexpressing cells by gel chromatography and that of disaccharide composition revealed that the CS chains had similar length and sulfation patterns. Furthermore, adenoviral gene delivery of the enzyme into intervertebral discs displayed a substantial increase in the level of CS biosynthesis. These observations indicate that CSGalNAcT-1 overexpression increases the number of CS chains attached to aggrecan core protein. Our studies may lead to a new therapeutic intervention, ameliorating the outcome of cartilage degenerative diseases.