Pentosan polysulfate binds to STRO-1+ mesenchymal progenitor cells, is internalized, and modifies gene expression: a novel approach of pre-programing stem cells for therapeutic application requiring their chondrogenesis.

BACKGROUND: The pharmaceutical agent pentosan polysulfate (PPS) is known to induce proliferation and chondrogenesis of mesenchymal progenitor cells (MPCs) in vitro and in vivo. However, the mechanism(s) of action of PPS in mediating these effects remains unresolved. In the present report we address this issue by investigating the binding and uptake of PPS by MPCs and monitoring gene expression and proteoglycan biosynthesis before and after the cells had been exposed to limited concentrations of PPS and then re-established in culture in the absence of the drug (MPC priming). METHODS: Immuno-selected STRO-1+ mesenchymal progenitor stem cells (MPCs) were prepared from human bone marrow aspirates and established in culture. The kinetics of uptake, shedding, and internalization of PPS by MPCs was determined by monitoring the concentration-dependent loss of PPS media concentrations using an enzyme-linked immunosorbent assay (ELISA) and the uptake of fluorescein isothiocyanate (FITC)-labelled PPS by MPCs. The proliferation of MPCs, following pre-incubation and removal of PPS (priming), was assessed using the Wst-8 assay method, and proteoglycan synthesis was determined by the incorporation of 35SO4 into their sulphated glycosaminoglycans. The changes in expression of MPC-related cell surface antigens of non-primed and PPS-primed MPCs from three donors was determined using flow cytometry. RNA sequencing of RNA isolated from non-primed and PPS-primed MPCs from the same donors was undertaken to identify the genes altered by the PPS priming protocol. RESULTS: The kinetic studies indicated that, in culture, PPS rapidly binds to MPC surface receptors, followed by internalisation and localization within the nucleus of the cells. Following PPS-priming of MPCs and a further 48 h of culture, both cell proliferation and proteoglycan synthesis were enhanced. Reduced expression of MPC-related cell surface antigen expression was promoted by the PPS priming, and RNA sequencing analysis revealed changes in the expression of 42 genes. CONCLUSION: This study has shown that priming of MPCs with low concentrations of PPS enhanced chondrogenesis and MPC proliferation by modifying their characteristic basal gene and protein expression. These findings offer a novel approach to re-programming mesenchymal stem cells for clinical indications which require the repair or regeneration of cartilaginous tissues such as in osteoarthritis and degenerative disc disease.

Characterization and flocculability of a novel proteoglycan produced by Talaromyces trachyspermus OU5.

A filamentous fungus strain OU5 was isolated from a soil sample for its ability to produce rich exopolymers (EPS), with high flocculation capability towards kaolin suspension and swine wastewater, at low-carbon source conditions. EPS from strain OU5 was extracted and characterized to determine its flocculating behavior and active constituents involved in the flocculation. Strain OU5 was identified as Talaromyces trachyspermus by 18S rDNA-ITS gene sequencing and morphological observation. The extracted EPS was a novel proteoglycan (designated as BF-OU5) composed of 84.6% (w/w) polysaccharides and 15.2% (w/w) proteins. The enzymatic digestion tests revealed that the polysaccharides in BF-OU5, composed of 67% glucose, 16.4% mannose, 8.6% xylose and 8% galactose, contributed to 99.7% of flocculating capacity and were the major active ingredients in the flocculation. By contrast, the proteins in BF-OU5 only had minor roles in the flocculation. The presence of hydroxyl, amide, carboxyl and methoxyl functional groups in BF-OU5, and the high molecular weight (1.053 × 10(5)-2.970 × 10(5) Da) as well as the structure of a spherical conformation with inner pores and channels made of cross-linked netted textures contributed to the flocculation. A dosage of 20 mg/l BF-OU5 initiated more than 92.5% of flocculating efficiency towards kaolin suspension without any added coagulants; its flocculability was stable over a wide range of pH (4.0-8.0) and temperature (20°C-100°C). Treatment of swine wastewater using BF-OU5 achieved 52.1% flocculating removal for chemical oxygen demand, 39.7% for Kjeldahl nitrogen, 18.6% for NH4(+)-N, 21.5% for total phosphorus, and 75% for turbidity.

Retinoic Acid Ameliorates Pancreatic Fibrosis and Inhibits the Activation of Pancreatic Stellate Cells in Mice with Experimental Chronic Pancreatitis via Suppressing the Wnt/ß-Catenin Signaling Pathway.

Pancreatic fibrosis, a prominent feature of chronic pancreatitis (CP), induces persistent and permanent damage in the pancreas. Pancreatic stellate cells (PSCs) provide a major source of extracellular matrix (ECM) deposition during pancreatic injury, and persistent activation of PSCs plays a vital role in the progression of pancreatic fibrosis. Retinoic acid (RA), a retinoid, has a broad range of biological functions, including regulation of cell differentiation and proliferation, attenuating progressive fibrosis of multiple organs. In the present study, we investigated the effects of RA on fibrosis in experimental CP and cultured PSCs. CP was induced in mice by repetitive cerulein injection in vivo, and mouse PSCs were isolated and activated in vitro. Suppression of pancreatic fibrosis upon administration of RA was confirmed based on reduction of histological damage, α-smooth muscle actin (α-SMA) expression and mRNA levels of ß-catenin, platelet-derived growth factor (PDGF)-Rß transforming growth factor (TGF)-ßRII and collagen 1α1 in vivo. Wnt 2 and ß-catenin protein levels were markedly down-regulated, while Axin 2 expression level was up-regulated in the presence of RA, both in vivo and in vitro. Nuclear translation of ß-catenin was significantly decreased following RA treatment, compared with cerulein-induced CP in mice and activated PSCs. Furthermore, RA induced significant PSC apoptosis, inhibited proliferation, suppressed TCF/LEF-dependent transcriptional activity and ECM production of PSC via down-regulation of TGFßRII, PDGFRß and collagen 1α1 in vitro. These results indicate a critical role of the Wnt/ß-catenin signaling pathway in RA-induced effects on CP and PSC regulation and support the potential of RA as a suppressor of pancreatic fibrosis in mice.

Effects of growth differentiation factor-9 and FSH on in vitro development, viability and mRNA expression in bovine preantral follicles.

The present study investigated the role of growth differentiation factor (GDF)-9 and FSH, alone or in combination, on the growth, viability and mRNA expression of FSH receptor, proliferating cell nuclear antigen (PCNA) and proteoglycan-related factors (i.e., hyaluronan synthase (HAS) 1, HAS2, versican, perlecan) in bovine secondary follicles before and after in vitro culture. After 12 days culture, sequential FSH (100 ng mL⁻¹) from Days 0 to 6 and 500 ng mL⁻¹ from Days 7 to 12) increased follicular diameter and resulted in increased antrum formation (P<0.05). Alone, 200 ng mL⁻¹ GDF-9 significantly reduced HAS1 mRNA levels, but increased versican and perlecan mRNA levels in whole follicles, which included the oocyte, theca and granulosa cells. Together, FSH and GDF-9 increased HAS2 and versican (VCAN) mRNA levels, but decreased PCNA mRNA expression, compared with levels in follicles cultured in α-minimum essential medium supplemented with 3.0 mg mL⁻¹ bovine serum albumin, 10 µg mL⁻¹ insulin, 5.5 µg mL⁻¹ transferrin, 5 ng mL⁻¹ selenium, 2 mM glutamine, 2mM hypoxanthine and 50 µg mL⁻¹ ascorbic acid (α-MEM⁺). Comparisons of uncultured (0.2 mm) and α-MEM⁺ cultured follicles revealed that HAS1 mRNA expression was higher, whereas VCAN expression was lower, in cultured follicles (P<0.05). Expression of HAS1, VCAN and perlecan (HSPG2) was higher in cultured than in vivo-grown (0.3 mm) follicles. In conclusion, FSH and/or GDF-9 promote follicular growth and antrum formation. Moreover, GDF-9 stimulates expression of versican and perlecan and interacts positively with FSH to increase HAS2 expression.

Characterizing the effects of VPA, VC and RCCS on rabbit keratocytes onto decellularized bovine cornea.

To investigate the morphological and growth characteristics of rabbit keratocytes when cultured on decellularized cornea under simulate microgravity (SMG) rotary cell culture system (RCCS) and static culture or in plastic culture supplemented with small molecules of valproic acid (VPA) and vitamin C (VC). Bovine corneas were firstly decellularized with Triton X-100 and NH(4)OH and through short-term freezing process. Then cell count kit-8 (CCK-8) and flow cytometry were used to test the effects of VPA and VC on the proliferation, cell cycle and apoptosis of rabbit keratocytes. Hematoxylin-eosin (H&E) staining and scanning electron microscopy (SEM) imaging showed that cells were eliminated in the decellularized bovine corneas. The proliferation of cultured keratocytes was promoted by VPA and VC in the cell proliferation assay. VPA and VC moderately decreased the number of apoptotic cells and obviously promoted cell-cycle entrance of keratocytes. Rabbit keratocytes in plastic displayed spindle shape and rare interconnected with or without VPA and VC. Cells revealed dendritic morphology and reticular cellular connections when cultured on the carriers of decellularized corneas supplemented with VPA and VC even in the presence of 10% fetal bovine serum (FBS). When cultured in RCCS supplemented with VPA, VC and 10% FBS, keratocytes displayed round shape with many prominences and were more prone to grow into the pores of carriers with aggregation. Reverse transcription-polymerase chain reaction (RT-PCR) analysis proved that the keratocytes cultured on decellularized bovine cornea under SMG with VPA and VC expressed keratocan and lumican. Keratocytes cultured on plastic expressed lumican but not keratocan. Immunofluorescence identification revealed that cells in all groups were positively immunostained for vimentin. Keratocytes on decellularized bovine cornea under SMG or in static culture were positively immunostained for keratocan and lumican. Thus, we reasonably made a conclusion that the combination of VPA, VC, RCCS and decellularized corneal carriers provide a good condition for keratocytes to well grow. Keratocytes can be manipulated to be aggregates or physiological morphological growth in vitro, which are important for the research of corneal stem cells and corneal tissue engineering.

IL-4 alone and in combination with IL-10 protects against blood-induced cartilage damage.

OBJECTIVE: It has been reported that interleukin (IL)-10 limits blood-induced cartilage damage. Our aim was to study the effect of IL-4 alone and in combination with IL-10 on blood-induced cartilage damage. DESIGN: Healthy human full thickness cartilage explants were cultured for 4 days in the presence of 50% v/v blood. IL-4, IL-10, or a combination of both cytokines was added during blood exposure. Cartilage matrix turnover was determined after a recovery period; additionally cytokine production, chondrocyte apoptosis, and expression of the IL-4 and IL-10 receptors were analyzed directly after exposure. RESULTS: Blood-induced damage to the cartilage matrix was limited by IL-4 in a dose-dependent way (P<0.05). Also IL-10 limited this damage, although to a lesser extent (P<0.03). The effect of IL-4 plus IL-10 was more pronounced and protective than IL-10 alone (P<0.05). Production of IL-1ß and tumor necrosis factor (TNF)-α was limited by both IL-4 and IL-10 (P<0.05), but more strongly by IL-4. Blood-induced apoptosis of chondrocytes was limited by IL-4 and the combination, and not by IL-10 alone. No direct beneficial effect of IL-4 or IL-10 on cartilage was found, however, the chondrocyte receptor expression of both cytokine receptors was upregulated by exposure to blood. CONCLUSIONS: This study demonstrates that IL-4 alone and in combination with IL-10 prevents blood-induced cartilage damage. Expectedly, anti-inflammatory effects on monocytes in the blood fraction and protective effects on chondrocytes are both involved. IL-4 in combination with IL-10 might be used to prevent blood-induced joint damage as a result of trauma or surgery.

The role of BMP-7 in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells in vitro.

This study addresses the role of bone morphogenetic protein-7 (BMP-7) in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells (BM MSCs) in vitro. BM MSCs were expanded and differentiated in the presence or absence of BMP-7 in monolayer and three-dimensional cultures. After 3 days of stimulation, BMP-7 significantly inhibited MSC growth in expansion cultures. When supplemented in commonly used induction media for 7-21 days, BMP-7 facilitated both chondrogenic and osteogenic differentiation of MSCs. This was evident by specific gene and protein expression analyses using real-time PCR, Western blot, histological, and immunohistochemical staining. BMP-7 supplementation appeared to enhance upregulation of lineage-specific markers, such as type II and type IX collagens (COL2A1, COL9A1) in chondrogenic and secreted phosphoprotein 1 (SPP1), osteocalcin (BGLAP), and osterix (SP7) in osteogenic differentiation. BMP-7 in the presence of TGF-beta3 induced superior chondrocytic proteoglycan accumulation, type II collagen, and SOX9 protein expression in alginate and pellet cultures compared to either factor alone. BMP-7 increased alkaline phosphatase activity and dose-dependently accelerated calcium mineralization of osteogenic differentiated MSCs. The potential of BMP-7 to promote adipogenesis of MSCs was restricted under osteogenic conditions, despite upregulation of adipocyte gene expression. These data suggest that BMP-7 is not a singular lineage determinant, rather it promotes both chondrogenic and osteogenic differentiation of MSCs by co-ordinating with initial lineage-specific signals to accelerate cell fate determination. BMP-7 may be a useful enhancer of in vitro differentiation of BM MSCs for cell-based tissue repair.

In vitro and in vivo modulation of cartilage degradation by a standardized Centella asiatica fraction.

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

BMP-13 emerges as a potential inhibitor of bone formation.

Bone morphogenetic protein-13 (BMP-13) plays an important role in skeletal development. In the light of a recent report that mutations in the BMP-13 gene are associated with spine vertebral fusion in Klippel-Feil syndrome, we hypothesized that BMP-13 signaling is crucial for regulating embryonic endochondral ossification. In this study, we found that BMP-13 inhibited the osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells (BM MSCs) in vitro. The endogenous BMP-13 gene expression in MSCs was examined under expansion conditions. The MSCs were then induced to differentiate into osteoblasts in osteo-inductive medium containing exogenous BMP-13. Gene expression was analysed by real-time PCR. Alkaline phosphatase (ALP) expression and activity, proteoglycan (PG) synthesis and matrix mineralization were assessed by cytological staining or ALP assay. Results showed that endogenous BMP-13 mRNA expression was higher than BMP-2 or -7 during MSC growth. BMP-13 supplementation strongly inhibited matrix mineralization and ALP activity of osteogenic differentiated MSCs, yet increased PG synthesis under the same conditions. In conclusion, BMP-13 inhibited osteogenic differentiation of MSCs, implying that functional mutations or deficiency of BMP-13 may allow excess bone formation. Our finding provides an insight into the molecular mechanisms and the therapeutic potential of BMP-13 in restricting pathological bone formation.

Modulation of xylosyltransferase I expression provides a mechanism regulating glycosaminoglycan chain synthesis during cartilage destruction and repair.

Osteoarthritis and rheumatoid arthritis are characterized by loss of proteoglycans (PGs) and their glycosaminoglycan (GAG) chains that are essential for cartilage function. Here, we investigated the role of glycosyltransferases (GTs) responsible for PG-GAG chain assembly during joint cartilage destruction and repair processes. At various times after antigen-induced arthritis (AIA) and papain-induced cartilage repair in rats, PG synthesis and deposition, expression of GTs, and GAG chain composition were analyzed. Our data showed that expression of the GT xylosyltransferase I (XT-I) gene initiating PG-GAG chain synthesis was significantly reduced in AIA rat cartilage and was associated with a decrease in PG synthesis. Interestingly, interleukin-1beta, the main proinflammatory cytokine incriminated in joint diseases, down-regulated the XT-I gene expression with a concomitant decrease in PG synthesis in rat cartilage explants ex vivo. However, cartilage from papain-injected rat knees showed up-regulation of XT-I gene expression and increased PG synthesis at early stages of cartilage repair, a process associated with up-regulation of TGF-beta1 gene expression and mediated by p38 mitogen-activated protein kinase activation. Consistently, silencing of XT-I expression by intraarticular injection of XT-I shRNA in rat knees prevented cartilage repair by decreasing PG synthesis and content. These findings show that GTs play a key role in the loss of PG-GAGs in joint diseases and identify novel targets for stimulating cartilage repair.

High levels of glucosamine-chondroitin sulfate can alter the cyclic preload and acute overload responses of chondral explants.

A recent study by our laboratory showed that 14 days of low intensity, intermittent cyclic preloading of chondral explants elevated the concentration of proteoglycans (PGs) to cause a mechanical stiffening of the explants prior to an acute overload and limit the extent of tissue damage. Longer term loading to 21 days resulted in tissue degradation prior to the acute traumatic event and excessive damage from an acute overload. Previous studies by others showed that bathing chondral explants in a supplement of glucosamine-chondroitin sulfate (glcN-CS) upregulated the synthesis of tissue PGs, particularly in stressed tissue, and the supplement served as an anti-inflammatory agent. Our current hypothesis was that the supplementation of culture media with a high concentration of glcN-CS would upregulate the production of tissue PG and limit or mitigate long-term degradation of chondral explants under cyclic preloading and limit tissue damage in an acute overload. We showed that, in the presence of supplement, cyclic preloading significantly increased tissue PG content and matrix modulus by about 65 and 300%, respectively, at 21 days, resulting in a reduction of matrix damage and cell death following an acute overload. These data show a biological action of high concentrations of this supplement and its effect on the mechanical properties in this in vitro model.

Low-level laser irradiation promotes cell proliferation and mRNA expression of type I collagen and decorin in porcine Achilles tendon fibroblasts in vitro.

Achilles tendon problems are commonly encountered in sports medicine and low-level laser therapy (LLLT) is widely used in rehabilitative applications to decrease pain, reduce inflammatory processes, and promote tissue healing. This study examined the effects on the proliferation of porcine Achilles tendon fibroblasts and gene expression, using different doses of low-level laser irradiation (LLLI). Four groups of identically cultured fibroblasts were exposed to LLLI and harvested after 24 h. The control group (Group 1) was subjected to no LLLI. Other groups received 1 J/cm2 (Group 2), 2 J/cm2 (Group 3), and 3 J/cm2 (Group 4), respectively. Cell proliferation and mRNA expressions of type I collagen and decorin were then measured. When compared to the control group, the cell proliferation of irradiated Achilles tendon fibroblasts in the other three groups increased significantly by 13% +/- 0.8% (Group 2), 30% +/- 0.4% (Group 3), and 12% +/- 0.6% (Group 4) respectively. But progressively higher laser intensity did not achieve a correspondingly higher cell proliferation effect in Achilles tendon fibroblasts. The mRNA expressions of decorin and type I collagen in fibroblasts with LLLI were significantly higher (p < 0.05). Therefore, suitable dosages of LLLI may result in more effective tissue healing by promoting type I collagen and decorin synthesis. However, these positive effects of LLLI on the repair of the Achilles tendon in humans should be further investigated in clinic.

Bone morphogenetic protein 7 (bmp-7) stimulates proteoglycan synthesis in human osteoarthritic chondrocytes in vitro.

BMP-7 is a member of the TGF-beta superfamily which is supposed to be one of the most potent anabolic factors of chondrocytes. In this study we analysed the effect of BMP-7 on three dimensional cultured chondrocytes with and without serum. Cartilage samples from fourteen patients with osteoarthritis of the knee were harvested and chondrocytes were cultivated in alginate-beads with and without serum supplementation (10% FCS). BMP-7 was added in three different concentrations (200, 600 and 1000 ng/ml). After 4 and 21 days PG concentration was determined by a Blyscan-Assay. For gene expression analysis of aggrecan (AGG) quantitative Lightcycler-PCR was used to estimate the mRNA levels. Under serumfree culture conditions there was no stimulation after 4 days but there was a twofold increase of PG concentration after 21 days. Using BMP-7 together with serum supplemented medium we found comparable results, however not as pronounced. AGG expression was increased only after 4 days but not after 21 days. Beside a stimulatory effect under serumfree conditions we also found a stimulatory effect of BMP-7 in the presence of serum. This study pronounces that BMP-7 might be a suitable anabolic activator of osteoarthritic chondrocytes.

Glucosamine supplementation accelerates early but not late atherosclerosis in LDL receptor-deficient mice.

Glucosamine, commonly consumed for the treatment of osteoarthritis, is classified as a nutritional supplement; however, there are few data regarding its metabolic or vascular effects. Glucosamine is a component of the hexosamine pathway, which has been implicated in the development of insulin resistance. Anecdotal reports suggest that glucosamine consumption can increase circulating cholesterol concentrations. To investigate the metabolic and vascular effects of glucosamine supplementation, we studied male and female LDL receptor-deficient mice fed a Western diet (21% fat, 0.15% cholesterol). Three groups of 6-10 mice of each gender received either no supplement, 15 mg . kg(-1) . d(-1) glucosamine (equivalent to an average human dose), or 50 mg . kg(-1) . d(-1) glucosamine added to their drinking water for 5, 10, or 20 wk. Plasma cholesterol and triglyceride concentrations increased in all mice with the addition of the Western diet. However, after 20 wk of treatment, cholesterol and triglyceride concentrations increased further in male mice consuming glucosamine compared with control groups. Glucosamine-supplemented mice had increased initiation of atherosclerosis after 5 wk; however, there was no effect on progression of atherosclerosis in either gender after longer periods of glucosamine supplementation (10 or 20 wk). Although long-term glucosamine supplementation exacerbated the hyperlipidemia in male mice, no increase in atherosclerosis occurred. Thus, glucosamine supplementation appears to be safe, with no adverse vascular consequences.

Arthritic disease suppression and cartilage protection with glycosaminoglycan polypeptide complexes (Peptacans) derived from the cartilage extracellular matrix: a novel approach to therapy.

Molecular fragments of cartilage are antigenic and can stimulate an autoimmune response. Oral administration of type II collagen prevents disease onset in animal models of arthritis but the effects of other matrix components have not been reported. We evaluated glycosaminoglycan polypeptides (GAG-P) and matrix proteins (CaP) from cartilage for a) mitigating disease activity in rats with collagen-induced arthritis (CIA) and adjuvant-induced arthritis (AIA) and b) stimulating proteoglycan (PG) synthesis by chondrocytes in-vitro. CIA and AIA were established in Wistar rats using standard methods. Agents were administered orally (10-200 mg/kg), either for seven days prior to disease induction (toleragenic protocol), or continuously for 15 days after injecting the arthritigen (prophylactic protocol). Joint swelling and arthritis scores were determined on day 15. Histological sections of joint tissues were assessed post-necropsy. In chondrocyte cultures, CaP + / - interleukin-1 stimulated PG biosynthesis. CaP was also active in preventing arthritis onset at 3.3, 10 or 20 mg/kg in the rat CIA model using the toleragenic protocol. It was only active at 20 and 200 mg/kg in the CIA prophylactic protocol. GAG-P was active in the CIA toleragenic protocol at 20 mg/kg but chondroitin sulfate and glucosamine hydrochloride or glucosamine sulfate were all inactive. The efficacy of CaP in the rat AIA model was less than in the CIA model. These findings lead us to suggest that oral CaP could be used as a disease-modifying anti-arthritic drug.

Oral administration of the NADPH-oxidase inhibitor apocynin partially restores diminished cartilage proteoglycan synthesis and reduces inflammation in mice.

Apocynin, an inhibitor of NADPH-oxidase, is known to partially reverse the inflammation-mediated cartilage proteoglycan synthesis in chondrocytes. More recently, it was reported that apocynin prevents cyclooxygenase (COX)-2 expression in monocytes. The present study aimed to investigate whether these in vitro features of apocynin could be confirmed in vivo. In a mouse model of zymosan-induced acute arthritis apocynin was administered orally (0, 3.2, 16 and 80 microg/ml in the drinking water) and the effects on cartilage proteoglycan synthesis were monitored. In a mouse model of zymosan-induced inflammation of the ears apocynin was administered orally (14 mg/kg/day by gavage) and the effects on ear swelling and ex vivo produced prostaglandin E2 (PGE2) by lipopolysaccharide (LPS)-stimulated blood cells were measured. In this study, ibuprofen was used as a positive control (50 mg/kg/day by gavage) and animals received vehicle as a negative control. Apocynin dose-dependently reversed the inhibition of proteoglycan synthesis in articular cartilage of the arthritic joint. A statistically significant increase in proteoglycan synthesis was found at a dose of 80 microg/ml apocynin. Apocynin did not affect the proteoglycan synthesis of the control knee joints. Apocynin significantly decreased the zymosan-induced ear swelling at 1, 2 and 4 h (hours) after zymosan injection versus the vehicle treated group at 14 mg/kg/day. The ex vivo production of PGE2 by LPS-stimulated blood cells was significantly decreased after in vivo apocynin treatment. Ibuprofen decreased ear swelling at the same time-points as apocynin and inhibited the ex vivo produced PGE2. In conclusion, the present study confirmed two important features of apocynin in vivo: (1) oral administration of apocynin can partially reverse the inflammation-induced inhibition of cartilage proteoglycan synthesis, and (2) oral administration of apocynin has COX inhibitory effects similar to the non-steroidal anti-inflammatory drug (NSAID) ibuprofen. Therefore, apocynin might be of potential use during the treatment of chronic inflammatory joint diseases like osteoarthritis or rheumatoid arthritis.

The potential of chitosan-based gels containing intervertebral disc cells for nucleus pulposus supplementation.

The suitability of chitosan-based hydrogels as scaffolds for the encapsulation of intervertebral disc (IVD) cells and the accumulation of a functional extracellular matrix mimicking that of the nucleus pulposus (NP) was investigated. The specific hypothesis under study was that the cationic chitosan would form an ideal environment in which large quantities of newly synthesized anionic proteoglycan could be entrapped. Indeed, all the formulations of cell-seeded chitosan hydrogels, studied under in vitro culture conditions, showed that the majority of proteoglycan produced by encapsulated NP cells was retained within the gel rather than released into the culture medium. This was not always the case when annulus fibrosus cells were encapsulated, as unlike the nucleus cells the annulus cells often did not survive when cultured in chitosan. The results support the concept that chitosan may be a suitable scaffold for cell-based supplementation to help restore the function of the NP during the early stages of IVD degeneration.

[Effects of lactone I from Atractylodes macrocephala Koidz on cytokines and proteolysis-inducing factors in cachectic cancer patients].

OBJECTIVE: To observe the effect of lactone I from Atractylodes macrocephala Koidz (LAMK I) on the cytokines and proteolysis-inducing factors (PIF) in cachectic cancer patients. METHOD: Sixty-four cachectic cancer patients were randomized into two groups, namely LAMK I group and FOE (fish oil-enriched nutritional supplementation) treatment group. The appetite, body weight changes, mid-arm muscle circumference (MAMC), and KPS scores were recorded 3 and 7 weeks after the treatment. Immunohistochemistry was used to analyze the changes in the cytokines interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF)-alpha and Western blotting employed to examine the changes of PIF after the treatment. RESULTS: The patients' appetite and MAMC in LAMKI group was better than those of patients in FOE group, and the serum IL-1 and TNF-alpha levels and urine PIF level were significantly lower than those of FOE group. Body weight and serum IL-6 level were not significantly different between the two groups. CONCLUSION: Lactone I from Atractylodes macrocephala Koidz can be beneficial for treating cancer cachexia.

Omega-3 polyunsaturated fatty acids regulate syndecan-1 expression in human breast cancer cells.

Human epidemiologic studies and animal model studies support a role for n-3 polyunsaturated fatty acids (n-3 PUFA) in prevention or inhibition of breast cancer. However, mechanisms for this protection remain unclear. Syndecan-1 is a heparan sulfate proteoglycan, expressed on the surface of mammary epithelial cells and known to regulate many biological processes, including cytoskeletal organization, growth factor signaling, and cell-cell adhesion. We studied effects of n-3 PUFA on syndecan-1 expression in human mammary cell lines. PUFA were delivered to cells by low-density lipoproteins (LDL) isolated from the plasma of monkeys fed diets enriched in fish oil (n-3 PUFA) or linoleic acid (n-6 PUFA). Proteoglycan synthesis was measured by incorporation of [35S]-sodium sulfate. No effect of either LDL was observed in nontumorigenic MCF-10A cells, whereas in MCF-7 breast cancer cells, treatment with n-3-enriched LDL but not n-6-enriched LDL resulted in significantly greater synthesis of a proteoglycan identified by immunoprecipitation as syndecan-1. Using real-time reverse transcription-PCR (RT-PCR), it was shown that n-3-enriched LDL significantly increased the expression of syndecan-1 mRNA in a dose-dependent manner and maximal effective time at 8 hours of treatment. The effect was mimicked by an agonist for peroxisome proliferator-activated receptor gamma (PPARgamma) and eliminated by the presence of PPARgamma antagonist suggesting a role for PPARgamma in syndecan enhancement. Our studies show that n-3 LDL modifies the production of syndecan-1 in human breast cancer cells and suggest that biological processes regulated by syndecan-1 may be modified through LDL delivery of n-3 PUFA.

Phenotypic analysis of bovine chondrocytes cultured in 3D collagen sponges: effect of serum substitutes.

Repair of damaged cartilage usually requires replacement tissue or substitute material. Tissue engineering is a promising means to produce replacement cartilage from autologous or allogeneic cell sources. Scaffolds provide a three-dimensional (3D) structure that is essential for chondrocyte function and synthesis of cartilage-specific matrix proteins (collagen type II, aggrecan) and sulfated proteoglycans. In this study, we assessed porous, 3D collagen sponges for in vitro engineering of cartilage in both standard and serum-free culture conditions. Bovine articular chondrocytes (bACs) cultured in 3D sponges accumulated and maintained cartilage matrix over 4 weeks, as assessed by quantitative measures of matrix content, synthesis, and gene expression. Chondrogenesis by bACs cultured with Nutridoma as a serum replacement was equivalent or better than control cultures in serum. In contrast, chondrogenesis in insulin-transferrin-selenium (ITS(+3)) serum replacement cultures was poor, apparently due to decreased cell survival. These data indicate that porous 3D collagen sponges maintain chondrocyte viability, shape, and synthetic activity by providing an environment favorable for high-density chondrogenesis. With quantitative assays for cartilage-specific gene expression and biochemical measures of chondrogenesis in these studies, we conclude that the collagen sponges have potential as a scaffold for cartilage tissue engineering.