Effects of transforming growth factor-beta on aggrecanase production and proteoglycan degradation by human chondrocytes in vitro

OBJECTIVE: Aggrecan is degraded by Aggrecanases (ADAMTS-4 and -5) and MMPs, which cleave its core protein at different sites. Transforming growth factor (TGF)beta is known to stimulate matrix formation in cartilage, and ADAMTS-4 production in synoviocytes. The aim of this in-vitro study was to examine the effects of TGFbeta on aggrecanase production in human cartilage. DESIGN: Expression of ADAMTS-4 and -5 in chondrocyte cultures from normal or osteoarthritic cartilage was studied at mRNA level by RT-PCR. Aggrecanase activity was examined by western blot of aggrecanase-generated neoepitope NITEGE, and by measure of proteoglycan degradation in cartilage explants. RESULTS: TGFbeta strongly increased mRNA levels of ADAMTS-4, while ADAMTS-5 was expressed in a constitutive way in chondrocytes from normal and osteoathritic cartilage. TGFbeta also increased NITEGE levels and proteoglycan degradation. Addition of an aggrecanase inhibitor blocked the increase of NITEGE, and partially inhibited proteoglycan degradation. CONCLUSIONS: TGFbeta stimulates ADAMTS-4 expression and aggrecan degradation in cartilage. This catabolic action seems to be partially mediated by aggrecanases. It is, therefore, proposed that the role of TGFbeta in cartilage matrix turnover is not limited to anabolic and anti-catabolic actions, but also extends to selective degradation of matrix components such as aggrecan.     

Enhanced matrix synthesis and in vitro formation of cartilage-like tissue by genetically modified chondrocytes expressing BMP-7.

Bone morphogenic protein-7 (BMP-7) supports ectopic cartilage and bone formation, is expressed in normal articular cartilage, and increases matrix synthesis in chondrocytes. Based on this knowledge, we hypothesized that an adenovirus (Ad) vector encoding human BMP-7 could be used to modify chondrocytes genetically to improve their capacity for cartilage repair. An adenovirus vector encoding BMP-7 (AdBMP-7) was constructed and its bioactivity confirmed by ectopic bone formation assay. AdBMP-7 modification of bovine chondrocytes induced expression of BMP-7 mRNA and bioactive protein, resulting in an increase in incorporation of 35SO4- into proteoglycan, 3H-proline uptake into protein, and the expression of the cartilage-specific matrix genes, aggrecan and type II collagen. An in vitro model of chondrocyte transplantation was used to demonstrate the feasibility of using genetically modified chondrocytes to enhance formation of cartilage-like tissue. When transplanted onto cartilage explants and maintained in vitro for 3 weeks, chondrocytes modified with AdBMP-7 formed 1.9-fold thicker tissue than chondrocytes modified with a control vector (P < 0.001). This tissue was positive for type II collagen and proteoglycan but negative for type X collagen and demonstrated a cartilage-like morphology. These observations suggest that Ad-mediated transfer of BMP-7 gene to chondrocytes enhances the chondrocyte-specific matrix synthesis and their capacity to form cartilage-like tissue, thus representing a strategy that may improve cell-based cartilage repair.     

BMP-2 induces the expression of chondrocyte-specific genes in bovine synovium-derived progenitor cells cultured in three-dimensional alginate hydrogel

OBJECTIVE: According to recent reports, the synovial membrane may contain mesenchymal stem cells with the potential to differentiate into chondrocytes under appropriate conditions. In order to assess the usefulness of synovium-derived progenitor cells for the purposes of cartilage tissue engineering, we explored their requirements for the expression of chondrocyte-specific genes after expansion in vitro. DESIGN: Mesenchymal progenitor cells were isolated from the synovial membranes of bovine shoulder joints and expanded in two-dimensions on plastic surfaces. They were then seeded either as micromass cultures or as single cells within alginate gels, which were cultured in serum-free medium. Under these three-dimensional conditions, chondrogenesis is known to be supported and maintained. Cell cultures were exposed either to bone morphogenetic protein-2 (BMP-2) or to isoforms of transforming growth factor-beta (TGF-beta). The levels of mRNA for Sox9, collagen types I and II and aggrecan were determined by RT-PCR. RESULTS: When transferred to alginate gel cultures, the fibroblast-like synovial cells assumed a rounded form. BMP-2, but not isoforms of TGF-beta, stimulated, in a dose-dependent manner, the production of messenger RNAs (mRNAs) for Sox9, type II collagen and aggrecan. Under optimal conditions, the expression levels of cartilage-specific genes were comparable to those within cultured articular cartilage chondrocytes. However, in contrast to cultured articular cartilage chondrocytes, synovial cells exposed to BMP-2 continued to express the mRNA for alpha1(I) collagen. CONCLUSIONS: This study demonstrates that bovine synovium-derived mesenchymal progenitor cells can be induced to express chondrocyte-specific genes. However, the differentiation process is not complete under the chosen conditions. The stimulation conditions required for full transformation must now be delineated.     

Osteogenic protein-1 promotes the synthesis and retention of extracellular matrix within bovine articular cartilage and chondrocyte cultures

OBJECTIVE: We have used recombinant osteogenic protein-1 to investigate our hypothesis that proper repair and maintenance of cartilage requires not only enhanced biosynthesis and replenishment of the extracellular matrix but also the enhancement of components necessary for matrix retention. DESIGN: The effects of osteogenic protein-1 were examined on bovine articular cartilage slices as well as isolated chondrocytes grown in alginate beads. Cartilage slices were examined for accumulation of proteoglycan by incorporation of 35S-sulfate and staining using Safranin O or, a biotinylated probe specific for hyaluronan. Bovine chondrocytes were characterized by use of a particle exclusion assay, in-situ hybridization, quantitative-competitive RT-PCR and a hyaluronan-binding assay. RESULTS: Osteogenic protein-1 treatment substantially enhanced the accumulation of hyaluronan and proteoglycan within cartilage tissue slices. As with the tissue, osteogenic protein-1 enhanced the size of cell-associated matrices assembled and retained by chondrocytes in vitro. This enhanced matrix assembly was paralleled by an increased expression of mRNA for aggrecan, hyaluronan synthase-2 and CD44. Of the two hyaluronan synthase genes expressed by chondrocytes, only hyaluronan synthase-2 was upregulated by osteogenic protein-1. Coupled with the increase in the CD44 mRNA was an increase in functional hyaluronan binding activity present at the chondrocyte cell surface. CONCLUSIONS: These results demonstrate that osteogenic protein-1 stimulates not only the synthesis of the major cartilage extracellular matrix component aggrecan, but also two associated molecules necessary for the retention of aggrecan, namely hyaluronan and CD44.     

Activation by IL-1 of bovine articular chondrocytes in culture within a 3D collagen-based scaffold. An in vitro model to address the effect of compounds with therapeutic potential in osteoarthritis

OBJECTIVE: To determine the best protocol for the preparation of a tissue-engineered cartilage to investigate the potential anti-arthritic and/or anti-osteoarthritic effects of drugs. METHODS: Calf articular chondrocytes, seeded in collagen sponges were grown in culture for up to 1 month. At day 14 cultures received interleukin (IL)-1beta (ranging from 0.1 to 20 ng/ml) for 1 to 3 days. Analyses of gene expression for extracellular matrix proteins, collagen-binding integrins, matrix metalloproteinases (MMPs), aggrecanases, TIMPs, IL-1Ra and Ikappa-Balpha were carried out using real-time polymerase chain reaction (PCR). Metalloproteinase activities were analysed in the culture medium using both zymography and fluorogenic peptide substrates. RESULTS: We selected a culture for 15 or 17 days with collagen sponges seeded with 10(7) chondrocytes showing a minimal cell proliferation, a maximal sulphated glycosaminoglycan (sGAG) deposition and a high expression of COL2A1, aggrecan and the alpha10 integrin sub-unit and low expression of COL1A2 and the alpha11 integrin sub-unit. In the presence of 1 ng/ml IL-1beta, we observed at day 15 up-regulations of 450-fold for MMP-1, 60-fold for MMP-13, 54-fold for ADAMTS-4 and MMP-3 and 10-fold for ADAMTS-5 and IL-1Ra. Down-regulations of 2.5-fold for COL2A1 and aggrecan were observed only at day 17. At the protein level a dose-dependent increase of total MMP-1 and MMP-13 was noted with less than 15% in the active form. CONCLUSIONS: This in vitro model of chondrocyte culture in three dimensional (3D) seems well adapted to investigate the responses of these cells to inflammatory cytokines and to evaluate the potential anti-inflammatory effects of drugs.     

Phenotypic stability of articular chondrocytes in vitro: the effects of culture models, bone morphogenetic protein 2, and serum supplementation.

Numerous in vitro culture models have been developed for the investigation of chondrocyte and cartilage biology. In this study, we investigated the stability of the chondrocytic phenotype in monolayer, aggregate, pellet, and explant culture models and assessed the effects of recombinant human bone morphogenetic protein 2 (rhBMP-2) and serum supplementation on the phenotype in each model. Phenotypic effects were assessed by analyses of procollagen type II, aggrecan, (V + C)- fibronectin, and procollagen type I messenger RNA expression. In monolayer cultures, we noted a characteristic loss of procollagen type II and induction of procollagen type I expression. The aggregate and pellet culture models supported matrix protein gene expression profiles more reflective of in vivo levels. In explant cultures, expression of matrix protein genes was consistently depressed. Treatment with rhBMP-2 significantly increased the expression of procollagen type II and aggrecan in monolayer cultures; however, other models showed comparatively little response. Similarly, serum supplementation significantly down-regulated procollagen type II and aggrecan expression in monolayer cultures but had less effect on gene expression in the other models. Serum supplementation increased procollagen type I expression in monolayer and aggregate cultures. These results suggest that the influence of exogenous BMP-2 and serum on expression of chondrocyte-specific matrix protein genes is influenced by aspects of substrate attachments, cellular morphology, and/or cytoskeletal organization. Finally, the analyses of fibronectin expression suggest that V and C region alternative splicing in chondrocytes is linked to the establishment of a three-dimensional multicellular complex.     

Fibronectin fragments upregulate insulin-like growth factor binding proteins in chondrocytes

Addition of fibronectin fragments (Fn-fs) to cultured cartilage explants has been shown to mediate extensive cartilage matrix degradation followed by anabolic responses. OBJECTIVE: To determine whether specific Fn-fs regulate cartilage metabolism through a mechanism, in part, involving insulin-like growth factor (IGF) and insulin-like growth factor binding proteins (IGFBPs). METHODS: Primary bovine articular chondrocyte cultures were treated with Fn-fs. mRNA from the cultures was analysed by Northern blotting. Changes in the levels of IGFBPs in cellular extracts and conditioned media were analysed by Western ligand blotting. Explant cultures of bovine articular cartilage were used to assay release of exogenous IGF-I and IGFBP-2. An analog of IGF-I with altered affinity for IGFBPs was used to assay the effect of IGFBPs on proteoglycan synthesis. RESULTS: The Fn-fs increased protein levels of IGFBPs-2, -3 and -5 in conditioned media and of IGFBP-2 in cell extracts by as much as nine-fold. Conversely, the protein level of constitutively expressed IGBP-4 was decreased in conditioned medium. Northern blot analysis reflected increased IGFBP-3 mRNA but not decreased IGFBP-4 mRNA. The IGF-I analog was more effective at restoring PG synthesis suppression by Fn-fs than was wild type IGF-I. CONCLUSIONS: The Fn-fs increased levels of IGFBPs in cultures of bovine articular chondrocytes and elicited release of IGFBP-2 and IGF-I from articular cartilage. The increased level of IGFBPs may trap IGF-I and account in part for the initial suppression of PG synthesis. Induced proteinases may subsequently liberate IGF-I and cause greatly enhanced anabolic processes, contributing to cartilage repair.     

AG-041R stimulates cartilage matrix synthesis without promoting terminal differentiation in rat articular chondrocytes

OBJECTIVE: AG-041R, a novel indolin-2-one derivative, has recently been demonstrated to induce systemic hyaline cartilage hyperplasia in rats. The aim of this study was to characterize its anabolic actions on chondrocytes. DESIGN: Chondrocytes were isolated from knee joints of 5-week-old SD rats. Effects of AG-041R on cartilage matrix synthesis were examined by measuring [(35)S]sulfate incorporation into proteoglycans, Alcian blue staining, and Northern blotting of cartilage matrix genes. ALP activity, mineral deposition and the expression of markers for hypertrophic chondrocytes, were assessed for terminal differentiation of chondrocytes. Roles of endogenous TGF-beta/BMPs and MEK1/Erk signaling in the action of AG-041R were investigated using the neutralizing soluble receptors and the MEK1 inhibitor. RESULTS: AG-041R accelerated proteoglycan synthesis assessed by both [(35)S]sulfate incorporation and Alcian blue stainable extracellular matrix accumulation. It also up-regulated the gene expression of type II collagen and aggrecan, as well as tenascin, a marker for articular cartilage. In contrast, AG-041R suppressed ALP activity, mineralization, and the gene expression of type X collagen and Cbfa1, indicating that AG-041R prevents chondrocyte terminal differentiation. AG-041R increased in BMP-2 mRNA, and the neutralizing soluble receptor for BMPs reversed the stimulatory effects of AG-041R on cartilage matrix synthesis. Moreover, AG-041R activated MEK1/Erk pathway, which was revealed to prevent chondrocyte terminal differentiation. CONCLUSION: AG-041R stimulates cartilage matrix synthesis without promoting terminal differentiation in rat articular chondrocytes, which is mediated at least in part by endogenous BMPs and Erk. The data demonstrates that AG-041R has a potential to be a useful therapeutic agent for articular cartilage disorders.     

Response of human chondrocytes prepared for autologous implantation to growth factors

This study investigated metabolism of autologous chondrocytes after initial expansion immediately before implantation. Chondrocytes cultured in either monolayers or alginate beads were treated with insulin-like growth factor-1 (IGF-1), osteogenic protein-1 (OP-1), or a combination. Proteoglycan synthesis and DNA content were tested in both cultures. Alginate beads also were analyzed with live/dead cell assay, safranin O/fast green stain for histology, and immunohistochemistry with antibodies against collagen type II and VI, aggrecan, decorin, and fibronectin. In monolayers, autologous chondrocytes changed their morphologic appearance. In alginate, they maintained chondrocytic phenotype. Growth factors, especially combined, promoted cell survival and induced chondrocyte proliferation. OP-1 stimulated the largest cartilage-specific matrix and the most accumulation of collagen type II and fibronectin, although the overall matrix synthesized by autologous chondrocyte implantation cells was smaller than that produced by normal chondrocytes. The clinical implications of this study suggest a significant promise for anabolic growth factors in cartilage repair as a potential modifying therapy for the enhancement of chondrocytic phenotype of autologous chondrocytes.     

Bone morphogenetic protein-7 antagonizes tumor necrosis factor-α–induced activation of nuclear factor κB and up-regulation of the ADAMTS,leading to decreased degradation of disc matrix macromolecules aggrecan and collagen II

Background contextTumor necrosis factor-α (TNF-α) is a regulatory cytokine that can increase the activity of enzymes such as ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs), which degrade disc matrix. ADAMTS are enzymes that break down disc matrix and thereby mediate disc degeneration. Bone morphogenetic protein-7 (BMP-7), on the other hand, stimulates synthesis of the disc extracellular matrix and is a potential therapeutic molecule for the treatment of disc degeneration. However, the effects of BMP-7 on TNF-α and ADAMTS are unknown.PurposeWe investigated the effects of BMP-7 on the catabolic regulators such as TNF-α and ADAMTS and evaluated the molecular mechanism by which BMP-7 affects the catabolic regulators.Study designThis was an in vitro study in which we used human intervertebral disc cells cultured in alginate beads.MethodsHuman intervertebral disc cells were cultured in alginate beads, and treated with TNF-α, or TNF- α plus BMP-7, pharmacological inhibitor of ERK1/2 (U0126), p38 (SB203580), or NFκB (BAY 11-7082). The mRNA levels of target genes were measured by real-time polymerase chain reaction, and the protein levels were determined by the Western blots. The nuclear factor (NF)κB activity was analyzed by measured phosphorylation and nuclear translocation of the NFκB protein p65.ResultsTNF-α activated NFκB signaling and induced up-regulation of the catabolic regulators ADAMTS-4 and ADAMTS-5, contributing to degradation of the disc matrix macromolecules aggrecan and collagen II. BMP-7 antagonized the TNF-α–induced activation of NFκB protein p65 and blocked TNF-α–induced up-regulation of ADAMTS-4 and ADAMTS-5, leading to reversing TNF-α–mediated degradation of aggrecan and collagen II. Moreover, BMP-7 antagonized the TNF-α–induced activation of NFκB signaling by suppressing phosphorylation and nucleus translocation of NFκB protein p65.ConclusionBMP-7 antagonizes TNF-α–induced activation of NFκB and up-regulation of ADAMTS, leading to decreased degradation of disc matrix macromolecules. These data indicate that BMP-7 has a dual mechanism of action on disc metabolism: (1) the previously well-described positive effect on disc matrix synthesis and (2) an anticatabolic effect that is described here. This understanding is important as BMP-7 is being considered for treatment of disc degeneration.     

Coordinate upregulation of cartilage matrix synthesis in fibrin cultures supplemented with exogenous insulin-like growth factor-I.

The addition of insulin-like growth factor-I to cartilage cultures is known to stimulate the synthesis of proteoglycan and type-II collagen in explant and monolayer studies. The purpose of this study was to determine the effects of long-term supplementation with insulin-like growth factor-I in chondrocytes cultured in fibrin discs as a preliminary investigation to in vivo application of chondrocyte/insulin-like growth factor-I/fibrin grafts to articular-cartilage repair procedures. Chondrocyte-fibrin cultures were maintained for 14 days, with insulin-like growth factor-I added at varying concentrations of 0, 10, 50, or 100 ng/ml medium. Cultures supplemented with 50 or 100 ng of growth factor/ml had increased levels of aggrecan and type-IIB procollagen mRNA, and translation to aggrecan and type-IIB collagen was confirmed by dye-binding assay of total proteoglycan, type-II collagen immunohistochemistry, and determination of collagen content by high-performance liquid chromatography. Maintenance of the chondrocyte phenotype during the 14 days of culture was confirmed by round cell morphology on routine staining, expression of type-II procollagen mRNA on in situ hybridization, evidence of production of pericellular type-II collagen on immunocytochemistry, synthesis of large-molecular-size aggrecan monomer on CL-2B column chromatography, and lack of appreciable message expression for type I or IIA collagen on Northern blot hybridization. Dose-response effects of insulin-like growth factor-I on the expression of chondrocyte matrix constituents were most pronounced at 50 and 100 ng of growth factor per milliliter of medium. These data confirm that (a) culture of chondrocytes for extended periods in three-dimensional cultures of fibrin maintains the chondrocyte phenotype and (b) supplementation with increasing concentrations of insulin-like growth factor-I enhances chondrocyte matrix synthesis and may provide a means to enhance chondrocyte phenotypic stability and function during transplantation grafting procedures.     

In vitro stimulation of articular chondrocyte mRNA and extracellular matrix synthesis by hydrostatic pressure

This study tested the effects of hydrostatic pressure (10 MPa) on adult articular chondrocyte mRNA and extracellular matrix synthesis in vitro. High density primary cultures of bovine chondrocytes were exposed to hydrostatic pressure applied intermittently at 1 Hz or constantly for 4 hours in serum-free medium or in medium containing 1% fetal bovine serum, mRNAs for aggrecan, types I and II collagen, and β-actin were analyzed by Northern blots and quantified by slot blots. Proteoglycan synthesis was quantified by ³⁵SO₄ uptake into cetylpyridinium chloride-precipitable glycosaminoglycans, and cell-associated aggrecan and type-II collagen were detected by immunohistochemical techniques. In serum-free medium, intermittent pressure increased aggrecan mRNA signal by 14% and constant pressure decreased type-II collagen mRNA signal by 16% (p < 0.05). In the presence of 1% fetal bovine serum, intermittent pressure increased aggrecan and type-II collagen mRNA signals by 31% (p < 0.01) and 36% (p < 0.001), respectively, whereas constant pressure had no effect on either mRNA. Intermittent and constant pressure stimulated glycosaminoglycan synthesis 65% (p < 0.001) and 32% (p < 0.05), respectively. Immunohistochemical detection of cell-associated aggrecan and type-II collagen was increased in response to both intermittent and constant pressure. These data support the hypothesis that physiologic hydrostatic pressure directly influences the extracellular matrix metabolism of articular chondrocytes.     

Recombinant human bone morphogenetic protein-2 maintains the articular chondrocyte phenotype in long-term culture

Bone morphogenetic proteins have been shown to increase matrix synthesis by articular chondrocytes in short-term cultures. Members of this family of proteins have also been shown to induce endochondral ossification in vivo. The present study was performed to determine if the addition of human recombinant bone morphogenetic protein-2 to a long-term monolayer articular chondrocyte cell culture system affected the ability of the chondrocytes to divide in vitro, whether the cytokine altered expression of the articular chondrocyte phenotype and synthesis of matrix proteoglycans, and whether the cytokine was capable of inducing differentiation to a hypertrophic chondrocyte. Human recombinant bone morphogenetic protein-2 did not alter cell proliferation. It caused 3.5–6.2 times more proteoglycan synthesis by articular chondrocytes during each of the time points tested after 4 days in culture. Total proteoglycan accumulation in the extracellular matrix after 28 days in culture was 6.7 times as great in the treated cultures as in the control. Treatment with human recombinant bone morphogenetic protein-2 maintained the articular chondrocyte phenotype of cells in culture as demonstrated by Northern blot analysis: the expression of type-I collagen genes was increased and that of type-II collagen and aggrecan mRNA was lost in untreated chondrocyte cultures after 14–21 days in culture. In contrast, exposure to 100 ng/ml human recombinant bone morphogenetic protein-2 maintained expression of type-II collagen and increased expression of aggrecan compared with controls during the 28-day culture period. Northern blot analysis of the expression of type-X collagen and osteocalcin by chondrocytes treated with human recombinant bone morphogenetic protein-2 showed a lack of expression of these genes, indicating no alteration in phenotype. These experiments demonstrated the ability of human recombinant bone morphogenetic protein-2 to promote the articular chondrocyte phenotype and matrix synthesis in long-term culture. Characteristics of cell growth were not affected, and the cytokine did not induce differentiation to a hypertrophic chondrocyte.     

Chondroitin sulphate impedes the migration of a sub-population of articular cartilage chondrocytes

OBJECTIVE: To determine whether chondroitin sulphate (CS) impedes the migration of primary articular chondrocytes. DESIGN: Articular chondrocytes were isolated from young and skeletally mature bovine animals. Boyden chambers were used to quantify chondrocyte migration on aggrecan in the presence and absence of CS chains. A novel in vitro model of cell migration into articular cartilage explants was designed to visualise and quantify the migration of labelled chondrocytes into cartilage matrix which had been treated with chondroitinase ABC to remove CS chains present. RESULTS: A consistent trend of increased migration with both age groups of a sub-population of chondrocytes was demonstrated on aggrecan in the absence of CS. These data were supported by results from the in vitro model of chondrocyte migration which demonstrated increasing numbers of a chondrocyte sub-population from both age groups of cartilage migrating into the chondroitinase ABC digested cartilage explants with time in culture. Minimal migration of these chondrocytes was demonstrated into phosphate buffered saline (PBS) treated control explants. CONCLUSIONS: We confirm that a sub-population of chondrocytes isolated from both young and skeletally mature articular cartilages have the ability to migrate. We also demonstrate that CS chains inhibit the migration of these articular chondrocytes and that their removal by chondroitinase ABC digestion enhances the migration of these chondrocytes. Such findings may provide a clinical application for improving cell-based cartilage repair strategies by enhancing integration between endogenous and repair tissue.     

Effect of dynamic compressive loading and its combination with a growth factor on the chondrocytic phenotype of 3-dimensional scaffold-embedded chondrocytes

Background and purpose Three-dimensionally (3D-) embedded chondrocytes have been suggested to maintain the chondrocytic phenotype. Furthermore, mechanical stress and growth factors have been found to be capable of enhancing cell proliferation and ECM synthesis. We investigated the effect of mechanical loading and growth factors on reactivation of the 3D-embedded chondrocytes.

Methods Freshly isolated chondrocytes from rat articular cartilage were grown in monolayer cultures and then in collagen gel. Real-time RT-PCR and histological analysis for aggrecan and type II and type I collagen was performed to evaluate their chondrocytic activity. Then, the 3D-embedded chondrocytes were cultured under either mechanical loading alone or in combination with growth factor. The dynamic compression (5% compression, 0.33 Hz) was loaded for 4 durations: 0, 10, 60, and 120 min/day. The growth factor administered was either basic fibroblast growth factor (bFGF) or bone morphogenetic protein-2 (BMP-2).

Results Mechanical loading statistically significantly reactivated the aggrecan and type II collagen expression with loading of 60 min/day as compared to the other durations. The presence of BMP-2 and bFGF clearly enhanced the aggrecan and type II collagen expression of 3D-embedded chondrocytes. Unlike previous reports using monolayer chondrocytes, however, BMP-2 or bFGF did not augment the chondrocytic phenotype when applied together with mechanical loading.

Interpretation Dynamic compression effectively reactivated the dedifferentiated chondrocytes in 3D culture. However, the growth factors did not play any synergistic role when applied with dynamic compressive loading, suggesting that growth factors should be administered at different time points during regeneration of the transplantation-ready cartilage.     

Cyclodextrin polysulphates repress IL-1 and promote the accumulation of chondrocyte extracellular matrix

OBJECTIVE: To evaluate the influence of cyclodextrin polysulphate (CDPS) on the extracellular matrix (ECM) metabolism of human articular cartilage chondrocytes. METHODS: Isolated chondrocytes from femoral condyle cartilage of human knee joints were cultured in gelled alginate to maintain their differentiated phenotype. During 1 week of culture, the cells were exposed to different concentrations of CDPS. Synthesis of aggrecans was investigated in these cultures after using Na(2)(35)SO(4) as a radioactive precursor during the last 24h of culture. The artificial matrix was then solubilised with Na-citrate and newly synthesised aggrecan aggregates, accumulated during culture, were liberated and assayed. The isolated chondrocytes were labelled with antibodies against aggrecan and type II collagen to analyse the ECM molecules in the cell-associated matrix (CAM). Plasma membrane levels of receptors for insulin-like growth factor-1 (IGF-1RI) and for interleukin-1 (IL-1RI and IL-1RII), as well as levels of IGF-1, IL-1alpha and -beta were determined after the cells had been permeabilized and stained with the appropriate antibodies. The release of IL-6 in the culture media was used as a variable reflecting auto/paracrine IL-1 activity of the cells in different experimental conditions. RESULTS: CDPS significantly increased total (35)S-incorporation rates in ECM aggrecan. When compared with controls, CDPS-treated chondrocytes expressed significantly higher CAM aggrecan and type II collagen levels. As plasma membrane-bound IGFR1 and intracellular IGF-1 levels remained unchanged, this increase in accumulated CAM compounds may have resulted from suppressed catabolic activities by the chondrocytes in culture. CDPS-treated cells expressed significantly lower amounts of intracellular IL-1alpha and -beta levels. Plasma membrane-bound IL-1RI and decoy IL-1RII remained unchanged. beta-cyclodextrin-treated chondrocytes released significantly less IL-6 in the supernatant culture media. CONCLUSION: CDPS is a novel polysulfated polysaccharide showing cartilage structure modifying effects in vitro as it improves the synthesis of aggrecan and the accumulation of CAM macromolecules. This effect probably resulted in part from the downregulation of IL-1.