Aggrecan mRNA Levels Research Articles

Effect of nitrogen-rich cell culture surfaces on type X collagen expression by bovine growth plate chondrocytes

BackgroundRecent evidence indicates that osteoarthritis (OA) may be a systemic disease since mesenchymal stem cells (MSCs) from OA patients express type X collagen, a marker of late stage chondrocyte hypertrophy (associated with endochondral ossification). We recently showed that the expression of type X collagen was suppressed when MSCs from OA patients were cultured on nitrogen (N)-rich plasma polymer layers, which we call "PPE:N" (N-doped plasma-polymerized ethylene, containing up to 36 atomic percentage (at.% ) of N.MethodsIn the present study, we examined the expression of type X collagen in fetal bovine growth plate chondrocytes (containing hypertrophic chondrocytes) cultured on PPE:N. We also studied the effect of PPE:N on the expression of matrix molecules such as type II collagen and aggrecan, as well as on proteases (matrix metalloproteinase-13 (MMP-13) and molecules implicated in cell division (cyclin B2). Two other culture surfaces, "hydrophilic" polystyrene (PS, regular culture dishes) and nitrogen-containing cation polystyrene (Primaria®), were also investigated for comparison.ResultsResults showed that type X collagen mRNA levels were suppressed when cultured for 4 days on PPE:N, suggesting that type X collagen is regulated similarly in hypertrophic chondrocytes and in human MSCs from OA patients. However, the levels of type X collagen mRNA almost returned to control value after 20 days in culture on these surfaces. Culture on the various surfaces had no significant effects on type II collagen, aggrecan, MMP-13, and cyclin B2 mRNA levels.ConclusionHypertrophy is diminished by culturing growth plate chondrocytes on nitrogen-rich surfaces, a mechanism that is beneficial for MSC chondrogenesis. Furthermore, one major advantage of such "intelligent surfaces" over recombinant growth factors for tissue engineering and cartilage repair is potentially large cost-saving.

Effect of Oxygen Levels on Proteoglycan Synthesis by Intervertebral Disc Cells

the response of cells from the annulus fibrosus (AF) and nucleus pulposus (NP) to varying oxygen (O2) concentrations was examined when cultured in alginate. to study the effect of O2 concentration on AF and NP cells. AF and NP cells possess different metabolic profiles in situ. However, it is not clear whether this difference is maintained in in vitro culture conditions. AF and NP cells can respond differently in the different systems, which may differ from the in vivo environment in terms of nutrient supply and O2 levels. In vivo, O2 levels vary from 1% to 5% within the intervertebral disc, and there is evidence that disc cell metabolism can vary with O2 concentrations. an alginate scaffold was seeded with bovine AF or NP cells and maintained in culture for up to 18 days under different O2 concentrations. The sulfated glycosaminoglycan (GAG) content in the culture medium and the expression of aggrecan, type I (COL1A2) and II (COL2A1) collagen genes were analyzed at day 9 and day 18. in both NP and AF cells cultured either in normoxia (21% O2) or in hypoxia (5% and 1% O2), the GAG content of the culture medium increased with time, though the rate of increase was diminished in 5% O2. With a decrease in O2 levels, the expression of aggrecan mRNA increased in NP cells. There was little effect of O2 on aggrecan mRNA level in AF cells. However, there was a slight decrease with time. Interestingly, aggrecan mRNA levels did not reflect GAG release for either NP or AF cells. There was no effect with time or O2 levels on COL2A1 message in NP cells. The highest Aggrecan/COL2 message ratio for NP cells was with 1% O2, suggesting this to be the best condition for maintaining the NP phenotype. COL1A2 gene expression in NP and AF cells increased with time, but showed little change with O2 levels in NP cells. The highest COL2/COL1 ratio in NP cells was also observed with 1% O2. Finally, NP cells tended to remain localized in the alginate beads, whereas AF cells tended to migrate from the beads. both NP and AF cells showed little change in GAG production with O2 levels ranging from 1% to 21%. Disc cell metabolism is not impaired at low O2 concentrations, which appear beneficial to matrix composition. Furthermore, low oxygen may promote a gelatinous NP matrix, whereas increased oxygen levels may promote a fibrous matrix.

S022023 RGD導入フィブロイン平面上に播種された軟骨細胞への低出力超音波パルス刺激の影響([S02202]細胞および分子のマイクロ・ナノスケール解析(2))

Several reports suggest that low-intensity pulsed ultrasound stimulation (LIPUS) facilitates chondrogenesis. Recently it has been suggested that LIPUS may be transmitted via Integrin. In this study, the Arg-Gly-Asp (RGD) amino acid sequence, which is a ligand of Integrin, was induced to the fibroin substrates by either gene transfer or physical mixing, and the variation of chondrocyte response to LIPUS was evaluated. Chondrocytes were seeded on three kinds of fibroin substrates: 1 wild-type, 2 transgenic and 3 mixed. LIPUS stimulation, with spatial and temporal average intensity of 30 mW/cm2, was applied to the chondrocytes at 12, 36, 60 hours and administered for 20 minutes each time. The results showed that GAG production, number of chondrocytes and mRNA levels of aggrecan, Sox 9 and Integrin ^ 1 was enhanced in the transgenic group by LIPUS administration, but these LIPUS-derived changes were not found in the wild-type and mixed groups. We previously reported that the adhesive force between chondrocytes and RGD transgenic fibroin surfaces was higher than that for mixed fibroin, suggesting that adhesive force is translated via RGD which bonds covalently to the fibroin proteins for the transgenic group. The present results suggest that the early biological adhesion via RGD on the transgenic fibroin is sensitive to LIPUS.

Snapshot of degenerative aging of porcine intervertebral disc: a model to unravel the molecular mechanisms

Larger animal models, such as porcine, have been validated as appropriate models of the human disc with respect to biomechanics and biochemistry. They are advantageous for research as the models are relatively straightforward to prepare and easily obtainable for research to perform surgical techniques. The intention of this study was to quantitatively analyze gene expression for collagen and proteoglycan components of the extracellular matrix and for collagenase (MMP-1) in porcine discs of varying ages (Newborn; 2-3weeks, Mature; 6-9 month, Older; 2-3 years). In this study, we observed that the cell number and GAG (glycosaminoglycan) formation dramatically decreased with aging. Also, gene expression in the annulus fibrosus (AF) and nucleus pulposus (NP) cells changed with aging. The level of MMP-1 mRNA increased with age and both type I, II collagens decreased with age. The level of aggrecan mRNA was highest in the mature group and decreased significantly with aging. In the mature group, MMP-1 expression was minimal compared to the newborn group. In AF cells, type II collagen was expressed at a high level in the mature group with a higher level of aggrecan, when aged NP showed a decrease in type II collagen. The model of IVD degeneration in the porcine disc shows many changes in gene expression with age that have been previously documented for human and may serve as a model for studying changes in IVD metabolism with age. We concluded that the porcine model is excellent to test hypotheses related to disc degeneration while permitting time-course study in biologically active systems.

061 ALTERED SCLEROSTIN SYNTHESIS IN CARTILAGE AND BONE CONTRIBUTE OA PATHOLOGY

Introduction: Canonical Wnt signalling and increased β-catenin activity have been implicated in the process of cartilage degradation in osteoarthritis (OA). Increased Wnt-induced signalling protein 1 (WISP1) in OA cartilage induces cartilage degradation by upregulating matrix metalloproteinases (MMPs) and aggrecanases (ADAMTS-4 and ADAMTS-5). Wnt signalling is inhibited by endogenous antagonists dickkopf-1 (DKK1), secreted frizzledrelated proteins (sFRP) and sclerostin (SOST). Despite increased chondrocyte β-catenin promoting cartilage degradation, insufficiency of sFRPB failed to alter OA cartilage damage in mice, and inhibition of DKK1 reduced rather than increased cartilage damage in a surgical model of OA in rats. These conflicting results demonstrate the complexity of Wnt-β-catenin regulation. SOST is a potent Wnt-β-catenin antagonist and a key regulator of bone metabolism but no previous studies have investigated its role in OA pathology. Methods: SOST, WISP-1 and β-catenin were immuno-localised in osteochondral sections of surgically-induced OA in sheep and mice, as well as human tibial plateaus obtained at arthroplasty for OA. Expression of Sost, Wisp-1, β-catenin, Lrp-5/6, Dkk1, Mmp-1 & -13, Adamts-4 & -5, Timp-1 & -3, aggrecan and collagen II by chondrocytes in OA, and with stimulation by 10ng/ml interleukin-1α (IL-1) or 100ng/ml tumor necrosis factor-α (TNF) in culture, was quantified by real-time RT-PCR. The effect of 25-500ng/ml rhSOST on cartilage degradation and chondrocyte gene expression was examined in explant culture. Results: Contrary to being osteocyte-specific, Sost mRNA was expressed by articular chondrocytes in all species, and was upregulated in OA in mice and by IL-1 but not TNF in culture. Chondrocyte SOST protein was significantly increased only in the focal area of cartilage damage in surgically-induced OA in sheep and mice, as well as end-stage human OA. In contrast, osteocyte SOST was focally decreased in the subchondral bone in OA in association with increased bone sclerosis. SOST was biologically active in chondrocytes, increasing expression of Mmp-13 from as early as 4 hours and maintained to 24 hours. At 48 hours, exogenous SOST decreased mRNA levels of aggrecan and type II collagen, inhibited Wnt-β-catenin signalling and down-regulated Mmp-13, Adamts-5 and Timp-1 and Timp-3 expression. SOST augmented IL-1’s stimulatory effects on chondrocyte Mmp-1, Adamts-4 and Adamts-5, and its inhibition of aggrecan and collagen II expression. Conclusions: Taken together, this data suggests that SOST may actively participate in the pathological progression of OA by affecting both cartilage and subchondral bone. The differential change in SOST in cartilage and subjacent bone highlight the potential tissue/cell specific regulation of Sost, and the opposing effects of altering Wnt-β-catenin activity in bone and cartilage. Overall the effects of SOST in cartilage are pro-catabolic, with inhibition of aggrecan, collagen II and Timp expression, and acute upregulation of Mmp-13. In bone, SOST increases osteoblast activity and bone formation, and inhibition of SOST and DKK1 have potential in treating osteoporosis. In light of this and the multiple potential mechanisms of action of SOST, it will be important to determine what effect its inhibition may have on progression of both bone and cartilage changes in OA in vivo.

Quantitative differences in intervertebral disc–matrix composition with age-related degeneration

This study was carried out to determine the effect of age on the intervertebral disc, using a rabbit model. Anulus fibrosus and nucleus pulposus tissue from New Zealand white rabbits aged 3 years old (old rabbits) and 6 months old (young rabbits) were used. The water content, the proteoglycan, the DNA content, and the mRNA levels of aggrecan, type I collagen, and type II collagen were all measured for each sample. Water, proteoglycan, DNA, and the mRNA levels of aggrecan and type II collagen were all greater in the nucleus pulposus of the young rabbits as compared to the old. For the anulus fibrosus, the difference between young and old is less marked with only proteoglycan and DNA being greater in the young disc as compared to the old. Clearly, according to our results, it is the nucleus pulposus that suffers the brunt of the changes with age.

Chondrogenic mRNA expression in prechondrogenic cells after blue laser irradiation

Low-level laser therapy (LLLT) has been used as a method for biostimulation. Cartilage develops through the differentiation of mesenchymal cells into chondrocytes, and differentiated chondrocytes in articular cartilage maintain cartilage homeostasis by synthesizing cartilage-specific extracellular matrix. The aim of this study is to evaluate the enhancement of chondrocyte differentiation and the expression levels of chondrogenic mRNA in prechondrogenic ATDC5 cells after laser irradiation. For chondrogenic induction, ATDC5 cells were irradiated with a blue laser (405 nm, continuous wave) at 100 mW/cm 2 for 180 s following incubation in chondrogenic differentiation medium. Differentiation after laser irradiation was quantitatively evaluated by the measurement of total collagen contents and chondrogenesis-related mRNAs. The total amount of collagen and mRNA levels of aggrecan, collagen type II, SOX-9, and DEC-1 were increased relative to those of a non-laser irradiated group after 14 days of laser irradiation. On the other hand, Ap-2α mRNA, a negative transcription factor of chondrogenesis, was dramatically decreased after laser irradiation. In addition, intracellular reactive oxygen species (ROS) were generated after laser irradiation. These results, for the first time, provide functional evidence that mRNA expression relating to chondrogenesis is increased, and Ap-2α is decreased immediately after laser irradiation. As this technique could readily be applied in situ to control the differentiation of cells at an implanted site within the body, this approach may have therapeutic potential for the restoration of damaged or diseased tissue.

Role of the low‐density lipoprotein receptor‐related protein‐1 in regulation of chondrocyte differentiation

The low-density lipoprotein receptor-related protein 1 (LRP1) is known as an endocytic and signal transmission receptor. We formerly reported the gene expression and the localization of LRP1 in cartilage tissue and chondrocytes, but its roles in the differentiation of chondrocytes remained to be investigated. Here, in order to address this issue, we employed RNAi strategy to knockdown lrp1 in chondrocytic cells and obtained findings indicating a critical role therein. As a result of lrp1 knockdown, aggrecan and col2a1 mRNA levels were decreased. However, that of col10a1 or mmp13 mRNA was rather increased. Under this condition, we performed a promoter assay for Axin2, which is known to be induced by activation of the WNT/beta-catenin (betacat) signaling pathway. Thereby, we found that Axin2 promoter activity was enhanced in the lrp1 knockdown cells. Furthermore, when the WNT/beta-catenin pathway was activated in chondrocytic cells by WNT3a or SB216763, which inhibits the phosphorylation of GSK3beta, the mRNA levels of aggrecan and col2a1 were decreased, whereas that of mmp13 was increased. Additionally, the level of phosphorylated protein kinase C (PKC) zeta was also decreased in the lrp1 knockdown cells. When the phosphorylation of PKCzeta was selectively inhibited, aggrecan and col2a1 mRNA levels decreased, whereas the mmp13 mRNA level increased. These data demonstrate that LRP1 exerts remarkable effects to retain the mature phenotype of chondrocytes as a critical mediator of cell signaling. Our findings also indicate that the onset of hypertrophy during endochondral ossification appears to be particularly dependent on the WNT and PKC signaling initiated by LRP1.

Stigmasterol: a phytosterol with potential anti-osteoarthritic properties

Although most studies have focused on the cholesterol-lowering activity of stigmasterol, other bioactivities have been ascribed to this plant sterol compound, one of which is a potential anti-inflammatory effect. To investigate the effects of stigmasterol, a plant sterol, on the inflammatory mediators and metalloproteinases produced by chondrocytes. We used a model of newborn mouse chondrocytes and human osteoarthritis (OA) chondrocytes in primary culture stimulated with or without IL-1beta (10 ng/ml), for 18 h. Cells were pre-incubated for 48 h with stigmasterol (20 microg/ml) compared to untreated cells. We initially investigated the presence of stigmasterol in chondrocyte, compared to other phytosterols. We then assessed the role of stigmasterol on the expression of various genes involved in inflammation (IL-6) and cartilage turn-over (MMP-3, -13, ADAMTS-4, -5, type II collagen, aggrecan) by quantitative Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). Additional experiments were carried out to monitor the production of MMP-3 and prostaglandin E2 (PGE(2)) by specific immuno-enzymatic assays. We eventually looked at the role of stigmasterol on NF-kappaB activation by western blot, using an anti-IkappaBalpha antibody. After 18 h of IL-1beta treatment, MMP-3, MMP-13, ADAMTS-4, but not ADAMTS-5 RNA expression were elevated, as well as MMP-3 and PGE(2) protein levels in mouse and human chondrocytes. Type II collagen and aggrecan mRNA levels were significatively reduced. Pre-incubation of stigmasterol to IL-1beta-treated cells significantly decreased these effects described above (significant reduction of MMP-3 mRNA in human and mouse, MMP-3 protein in mouse, MMP-13 mRNA in mouse and human, ADAMTS-4 mRNA in human, PGE(2) protein in human and mouse) Finally, stigmasterol was capable of counteracting the IL-1beta-induced NF-kappaB pathway. This study shows that stigmasterol inhibits several pro-inflammatory and matrix degradation mediators typically involved in OA-induced cartilage degradation, at least in part through the inhibition of the NF-kappaB pathway. These promising results justify further ex vivo and in vivo investigations with stigmasterol.

RGD-dependent integrins are mechanotransducers in dynamically compressed tissue-engineered cartilage constructs

Recent studies have shown that integrins act as mechanoreceptors in articular cartilage. In this study, we examined the effect of blocking RGD-dependent integrins on both ECM gene expression and ECM protein synthesis. Chondrocytes were isolated from full-depth porcine articular cartilage and seeded in 3% agarose constructs. These constructs were loaded in compression with 15% strain at 0.33 and 1 Hz for 12 h, in the presence or absence of GRGDSP, which blocks RGD-dependent integrin receptors. The levels of mRNA for aggrecan, collagen II and MMP-3 were determined by semi-quantitative PCR at several time points up to 24 h post-stimulation. DNA and sGAG content were determined at several time points up to 28 days post-stimulation. At 0.33 Hz, the mRNA levels for aggrecan and MMP-3 were increased after loading, but the mRNA levels for collagen II remained unchanged. Incubation with GRGDSP counteracted these effects. Loading at 1 Hz led to increased mRNA levels for all three molecules directly after loading and these effects were counteracted by incubation with GRGDSP. The constructs that were loaded at 0.33 Hz showed a lower amount of sGAG, compared to the unstrained control. In contrast, loading at 1 Hz caused an increase in sGAG deposition over the culture period. Blocking integrins had only a counteracting effect on the long-term biosynthetic response of constructs that were compressed at 1 Hz. The results confirmed the role of RGD-dependent integrins as mechanotransducers in the regulation of both ECM gene expression and matrix biosynthesis for chondrocytes seeded in agarose under the applied loading regime. Interestingly, this role seems to be dependent on the applied loading frequency.

Chondroprotective effect of the bioactive peptide prolyl-hydroxyproline in mouse articular cartilage in vitro and in vivo

To investigate the direct effect of prolyl-hydroxyproline (Pro-Hyp) on chondrocytes under in vivo and in vitro conditions in an attempt to identify Pro-Hyp as the bioactive peptide in collagen hydrolysate (CH). The in vivo effects of CH and Pro-Hyp intake on articular cartilage were studied by microscopic examination of sections of dissected articular cartilage from treated C57BL/6J mice. In this study, mice that were fed diets containing excess phosphorus were used as an in vivo model. This mouse line showed loss of chondrocytes and reduced thickness of articular cartilage, with abnormality of the subchondral bone. The in vitro effects of CH, Pro-Hyp, amino acids and other peptides on proliferation, differentiation, glycosaminoglycan content and mineralization of chondrocytes were determined by MTT activity and staining with alkaline phosphatase, alcian blue and alizarin red. Expression of chondrogenesis-specific genes in ATDC5 cells was determined by semiquantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR). In vivo, CH and Pro-Hyp inhibited the loss of chondrocytes and thinning of the articular cartilage layer caused by phosphorus-induced degradation. In the in vitro study, CH and Pro-Hyp did not affect chondrocyte proliferation but inhibited their differentiation into mineralized chondrocytes. A combination of amino acids such as proline, hydroxyproline and prolyl-hydroxyprolyl-glycine did not affect chondrocyte proliferation or differentiation. Moreover, CH and Pro-Hyp caused two and threefold increases, respectively, in the staining area of glycosaminoglycan in the extracellular matrix of ATDC5 cells. RT-PCR indicated that Pro-Hyp increased the aggrecan mRNA level approximately twofold and decreased the Runx1 and osteocalcin mRNA levels by two-thirds and one-tenth, respectively. Pro-Hyp is the first bioactive edible peptide derived from CH to be shown to affect chondrocyte differentiation under pathological conditions.

Effects of Alendronate on A Disintegrin and Metalloproteinase with Thrombospondin Motifs Expression in the Developing Epiphyseal Cartilage in Rats

A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) have been reported to play a role in the degradation of aggrecan, a major component of cartilage. This study was performed to examine the effects of alendronate on the expression of ADAMTS in developing femoral epiphyseal cartilage. Primary cultured chondrocytes from this cartilage were treated with alendronate in vitro and postnatal day 1 rats were injected subcutaneously with alendronate (1 mg/kg) every second day in vivo. The number of cultured chondrocytes and their aggrecan mRNA levels were unaffected by the alendronate treatment at 10(-6) to 10(-4) M concentrations. The mRNA levels of ADAMTS-1, -2 and -9 in chondrocytes were also unaffected. However, the levels of ADAMTS-5 and -4 were reduced significantly by the same treatment. The thickness of the proliferating chondrocyte layers and the aggrecan mRNA levels in the epiphysis were unaffected by the alendronate treatment in vivo. However, the hypertrophied chondrocyte layers became significantly thicker, and the size of the secondary ossification centre was reduced significantly by the same treatment (P < 0.05). Both ADAMTS-4 and -5 mRNA expressions were also reduced significantly in vivo. The immunoreactivity against ADAMTS-4 was seen in hypertrophied chondrocytes and reduced significantly by the alendronate treatment. These results suggested that alendronate can inhibit the degradation of aggrecan in the articular cartilage by downregulating the expression of matrix enzymes such as ADAMTS-4 and -5.

Effect of Cytoskeletal Disruption on Mechanotransduction of Hydrostatic Pressure by C3H10T1/2 Murine Fibroblasts

Cyclic hydrostatic pressure of physiological magnitude (< 10 MPa) stimulates chondrogenic differentiation of mesenchymal stem cells, but mechanotransduction mechanisms are not well understood. It was hypothesized that an intact cytoskeleton would be required for uninhibited mechanotransduction of hydrostatic pressure. Therefore we examined the effects of drugs which selectively interfere with actin and tubulin polymerization on pressure-induced upregulation of aggrecan and col2a1 (type II collagen) mRNA expression. C3H10T1/2 cells were cultured as pellets in either 4µM cytochalasin D or 4µM nocodazole and subjected to 3 days of cyclic hydrostatic compression (1 Hz, 5 MPa, 2 h per day). Phalloidin staining and indirect immunostaining with anti α-tubulin antibody confirmed disruption of microfilament and microtubule assemblies, respectively. Real time RT-PCR revealed that both drugs substantially lowered the basal level of aggrecan and col2a1 mRNA, but that neither drug prevented a pressure-stimulated increase in gene expression relative to the altered basal state. Thus upregulation of macromolecular gene expression by cyclic hydrostatic pressure did not require a completely intact cytoskeleton.

The alpha5beta1 integrin as mechanotransducer in chondrocytes

Mechanical loading has been proposed as a mechanism for improving the functionality of engineered cartilage tissue. However, the precise mechanotransduction pathways are unknown. Recent studies have shown that integrins can act as mechanoreceptors in articular cartilage. In this study, we examined the role of integrin a5s1 in the anoregulation of both ECM gene expression and ECM protein synthesis in tissue-engineered hondrocyte/agarose constructs. Chondrocytes, isolated and seeded in 3% agarose constructs, were compressed at 0.33 and 1 Hz, in the presence or absence of GRGDSP (a blocker for integrin a5s1). mRNA levels for aggrecan, collagen II and MMP-3 were determined at several time points up to 24 hours post-stimulation. Cell viability, DNA and sGAG content were determined at several time points up to 28 days post-stimulation. mRNA levels for all genes were upregulated upon loading, except for collagen II, loaded at 0.33 Hz. Incubation with GRGDSP counteracted upregulation. sGAG levels were significantly lower in constructs loaded at 0.33 Hz compared to the unstrained control at day 28. In contrast, loading at 1 Hz caused a significant increase in sGAG deposition at this timepoint, which was counteracted by blocking the a5s1 integrin. We conclude that the a5s1 integrin acts as a mechanotransducer in the regulation of both ECM gene expression and matrix biosynthesis for chondrocytes seeded in agarose under the loading regimes applied. However, this regulation appears frequency dependent.

A10 TRANSFORMING GROWTH FACTOR-BETA INDUCES PERSISTENT SYNOVIAL FIBROSIS WHILE CONNECTIVE TISSUE GROWTH FACTOR-INDUCED FIBROSIS IS REVERSIBLE. ROLE FOR LYSYS HYDROXYLASE

Purpose: The main characteristics of osteoarthritis are cartilage damage, osteophyte formation and synovial fibrosis. Synovial fibrosis can contribute to the loss of the function of an OA joint. TGF-b is considered one of the main players in fibrotic diseases. However, CTGF, which can be induced by TGF-b, has been reported to be an important player as well, stated to contribute to maintenance of fibrotic tissue. We investigated the underlying mechanism in that leads to the observed difference in fibrosis persistence between TGF-band CTGF. Methods: We injected C57Bl/6 mice intra-articularly with Ad-TGF-b or Ad-CTGF. After 3, 7 and 21 days knee joint synovial tissue was isolated for RNA isolation or whole knee joints for histology. With Q-RT-PCR relative mRNA levels were determined of matrix molecules, proteases, protease inhibitors, and growth factors. To analyze the enzymes involved in collagen cross link formation, mRNA expression of lysyl hydroxylase (LH) was determined. CTGF-levels induced by TGF-b were measured by ELISA in 24 hour patella-synovial wash-outs of C57Bl/6 mice intra-articularly injected with Ad-TGF-b (3, 7 and 14 days). Results: TGF-b and CTGF induced synovial fibrosis in murine knee joints as shown by histology (as observed by an increase in synovial width). However, TGF-b-induced fibrosis was very persistent, whereas CTGFinduced fibrosis resorbed by day 28. TGF-b induced elevated collagen type I and very high levels of aggrecan expression and hardly any changes in collagen type II and III. CTGF induced no clear changes in collagen type I, II and III. CTGF even decreased aggrecan expression (day 21 only). CTGF induced no changes in MMP3, -9, -13, ADAMTS-4 or -5, whereas TGF-b increased MMP-3, -13 and ADAMTS4. Thus elevated levels of matrix degrading enzyme expression cannot explain the less persistent CTGF-induced fibrosis. TIMP1 (associated with matrix accumulation in fibrotic disorders), was highly up regulated by TGF-b (all days) and only slightly up regulated by CTGF (day 7). TGF-b also induced elevated mRNA levels of TGF-b1 and CTGF. Only TGF-b induced high levels of LH expression, especially LH2b, which has been implicated in hard-to-degrade collagen linkage and was found up regulated in fibrotic lesions. As TGF-b can induce CTGF and up regulated CTGF mRNA in our experiments, we measured CTGF levels in 24 hour patella-synovial wash outs and found that TGF-b induced 64 ng CTGF/ml after 3 days and that CTGF levels were still elevated by day 14. Conclusions: TGF-b induced increased collagen type I and aggrecan mRNA levels in synovial tissue, but also high levels of degrading enzymes. The latter does not explain the persistent nature of TGF-b versus CTGFinduced synovial fibrosis. Lack of collagen type I mRNA induced by CTGF corresponds to findings of other groups. Despite this fact, fibrosis is found. Only TGF-b induced high levels of TIMP1 expression, which was previously found associated with strain dependent fibrosis sensitivity in mice and implicated in accumulation of ECM in fibrotic disorders. TGF-b, not CTGF, induced high levels of LH which are crucial for ECM cross linking. Moreover, LH2b was reported by van der Slot et al. to induce harder-to-degrade crosslinks compared to LH2a. The 2b splicing variant was highly expressed upon stimulation with TGF-b. Since TGF-b clearly up regulated MMP expression a dominant role for TIMP appears unlikely. The strong induction of LH2b by TGF-b compared to CTGF, and consequently harder to degrade cross links, appears to be the most likely cause of the induction of irreversible fibrosis by TGF-b. A11 EFFICACY OF A SINGLE ULTRASOUND GUIDED INJECTION IN HIP OSTEOARTHRITIS

Differential regulation of chondroitin sulfate proteoglycan mRNAs in the denervated rat fascia dentata after unilateral entorhinal cortex lesion

Following brain trauma, chondroitin sulphate proteoglycans (CSPGs) are enriched at injury sites and in denervated areas. At injury sites, CSPGs are regarded as inhibitors of axonal regeneration because of their growth inhibitory properties. In areas of denervation their role is less clear, since they are enriched in zones of sprouting, i.e. zones of axonal growth. To identify CSPGs expressed in a denervated brain area and to quantify changes in their mRNA expression, neurocan, brevican, NG2, phosphacan and aggrecan mRNA were analyzed in the rat fascia dentata following entorhinal denervation. Laser microdissection was combined with quantitative RT-PCR to measure mRNA changes specifically within the denervated portion of the molecular layer (1h, 6h, 10h, 12h, 1d, 2d, 3d, 4d, 7d and 14d post-lesion). Changes in glial fibrillary protein mRNA were measured at the same time points and used as lesion control. This approach revealed a differential regulation of CSPG mRNAs in the denervated zone: neurocan, brevican and NG2 mRNA were upregulated with a maximum around 2 days post-lesion. In contrast, aggrecan mRNA levels reached a maximum 7 days post-lesion and phosphacan mRNA levels were not significantly altered. Taken together, our data reveal a temporal pattern in CSPG mRNA expression in the denervated fascia dentata. This suggests specific biological functions for CSPGs during the denervation-induced reorganization process: whereas the early increase in CSPGs in the denervated zone could influence the pattern of sprouting, the late increase of aggrecan mRNA suggests a different role during the late phase of reorganization.

Static Compression of Single Chondrocytes Catabolically Modifies Single-Cell Gene Expression

Previous work has established that mechanical forces can lead to quantifiable alterations in cell function. However, how forces change gene expression in a single cell and the mechanisms of force transmission to the nucleus are poorly understood. Here we demonstrate that the gene expression of proteins related to the extracellular matrix in single articular chondrocytes is modified by compressive forces in a dosage-dependent manner. Increasing force exposure catabolically shifts single-cell mRNA levels of aggrecan, collagen IIa, and tissue inhibitor of metalloproteinase-1. Cytohistochemistry reveals that the majority of strain experienced by the cell is also experienced by the nucleus, resulting in considerable changes in nuclear volume and structure. Transforming growth factor- β1 and insulin-like growth factor-I offer mechanoprotection and recovery of gene expression of aggrecan and metalloproteinase-1. These results suggest that forces directly influence gene transcription and may do so by changing chromatin conformation.

Anti-inflammatory effect of antidiabetic thiazolidinediones prevents bone resorption rather than cartilage changes in experimental polyarthritis

BackgroundRosiglitazone and pioglitazone are high-affinity peroxisome proliferator-activated receptor (PPAR)-γ agonists with potent anti-diabetic properties and potential anti-inflammatory effects. We compared the ability of a range of oral doses of these thiazolidinediones, including those sufficient to restore insulin sensitization, to inhibit the pathogenesis of adjuvant-induced arthritis (AIA).MethodsAIA was induced in Lewis rats by a subcutaneous injection of 1 mg of complete Freund's adjuvant. Rats were treated orally for 21 days with pioglitazone 3, 10 or 30 mg/kg/day, rosiglitazone 3 or 10 mg/kg/day, or with vehicle only. The time course of AIA was evaluated by biotelemetry to monitor body temperature and locomotor activity, by clinical score and plethysmographic measurement of hindpaw oedema. At necropsy, RT-PCR analysis was performed on synovium, liver and subcutaneous fat. Changes in cartilage were evaluated by histological examination of ankle joints, radiolabelled sulphate incorporation (proteoglycan synthesis), glycosaminoglycan content (proteoglycan turnover) and aggrecan expression in patellar cartilage. Whole-body bone mineral content was measured by dual-energy X-ray absorptiometry.ResultsThe highest doses of rosiglitazone (10 mg/kg/day) or pioglitazone (30 mg/kg/day) were required to reduce fever peaks associated with acute or chronic inflammation, respectively, and to decrease arthritis severity. At these doses, thiazolidinediones reduced synovitis and synovial expression of TNF-α, IL-1β and basic fibroblast growth factor without affecting neovascularization or the expression of vascular endothelial growth factor. Thiazolidinediones failed to prevent cartilage lesions and arthritis-induced inhibition of proteoglycan synthesis, aggrecan mRNA level or glycosaminoglycan content in patellar cartilage, but reduced bone erosions and inflammatory bone loss. A trend towards lower urinary levels of deoxipyridinolin was also noted in arthritic rats treated with thiazolidinediones. Rosiglitazone 10 mg/kg/day or pioglitazone 30 mg/kg/day increased the expression of PPAR-γ and adiponectin in adipose tissue, confirming that they were activating PPAR-γ in inflammatory conditions, although an increase in fat mass percentage was observed for the most anti-arthritic dose.ConclusionThese data emphasize that higher dosages of thiazolidinediones are required for the treatment of arthritis than for restoring insulin sensitivity but that thiazolidinediones prevent inflammatory bone loss despite exposing animals to increased fatness possibly resulting from excessive activation of PPAR-γ.

CD47 associates with alpha 5 integrin and regulates responses of human articular chondrocytes to mechanical stimulation in an in vitromodel

BackgroundRecent studies provide evidence of roles for integrins in mechanical signalling in bone and cartilage. Integrin signalling is modulated by various mechanisms, including interaction with other transmembrane proteins. We aimed to identify whether one such protein, integrin-associated protein (CD47/IAP), is expressed by chondrocytes and whether it may regulate integrin-dependent mechanotransduction.MethodsChondrocytes, isolated from macroscopically normal and osteoarthritic articular cartilage of human knee joints, were studied in a resting state or following mechanical stimulation at 0.33 Hz. CD47/IAP expression and associations were confirmed by immunohistology, reverse transcription-polymerase chain reaction, Western blotting, and immunoprecipitation. Roles in mechanotransduction were studied by assessing effects of function-blocking antibodies on a range of electrophysiological, cellular, and molecular responses of primary chondrocytes and responses of CD47/IAP-null cell lines transfected with CD47/IAP.ResultsHuman articular chondrocytes were shown to express CD47/IAP, predominantly the type 2 isoform. Immunoprecipitation showed association of CD47/IAP with α5 integrin and thrombospondin but not SIRPα (signal-regulatory protein-alpha). The function-blocking anti-CD47/IAP antibody Bric 126 inhibited changes in membrane potential, tyrosine phosphorylation, and elevation of relative levels of aggrecan mRNA induced by mechanical stimulation, whereas in the presence of B6H12, an antibody that has partial agonist activity, a membrane depolarisation rather than a membrane hyperpolarisation response was induced by mechanical stimulation. CD47-null cell lines did not show changes in cell membrane potential following mechanical stimulation. Changes in cell membrane potential following mechanical stimulation were seen when CD47-null cells were transfected with CD47/IAP expression vectors but were not seen following mechanical stimulation of cells transfected with vectors for the extracellular immunoglobulin variable (IgV) domain of CD47/IAP in the absence of the transmembrane and intracellular domains.ConclusionCD47/IAP is necessary for chondrocyte mechanotransduction. Through interactions with α5β1 integrin and thrombospondin, CD47/IAP may modulate chondrocyte responses to mechanical signals.

Chondrocyte-associated antigen and matrix components in a 2- and 3-dimensional culture of rat chondrocytes

The goal of this study was to compare expression of chondrocyte-associated antigen (CAA) and cartilage matrix molecules in 2-D (monolayer) and 3-D (Matrigel) culture. Chondrocytes isolated from the cartilage of 3-day-old rats were expanded in monolayer culture for 28 days. CAA expression gradually decreased and was not detected beyond the 96th hour of monolayer culture. Collagen type II and aggrecan mRNA levels decreased during culture. The collagen type I mRNA level increased during the first week and remained high. The increase in the versican mRNA level was less pronounced during the first week and declined slightly after further cultivation. Freshly isolated chondrocytes introduced into Matrigel still expressed CAA after 7 days. Moreover, CAA expression returned in chondrocytes re-cultured in Matrigel after 7 days in monolayer. Similarly, the increase in the mRNA levels of collagen type II and aggrecan in Matrigel was limited to freshly isolated chondrocytes and to those that remained in monolayer for 1 week. Collagen type I mRNA in monolayer and Matrigel cultures of freshly isolated chondrocytes was at a similar level. The introduction of freshly isolated or 7-day monolayer-cultured chondrocytes into Matrigel caused a decrease in the versican mRNA level in comparison with 7- and 14-day 2-D cultures, respectively. On the other hand, chondrocytes seeded in Matrigel after 14 days of monolayer culture did not express CAA, showed decreased levels of collagen type II and aggrecan mRNA and an increase in versican mRNA. In conclusion, it appears that changes in the expression of CAA in 2- and 3-D cultures occur in parallel to changes in typical cartilage matrix molecule expression.