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With the aim to increase type II collagen content in the scaffold-free cartilage-like cell sheet using human bone marrow mesenchymal stem cells, we examined the effect of epigallocatechin-3-gallate (EGCG) addition to the chondrogenic medium for the cell sheet culture. The addition of EGCG (10 μM) increased the content of type II collagen 2-fold, while the addition did not markedly change the expression level of the genes encoding type II collagen and Sox 9. The reactive oxygen species level in the cells in cell sheets was thought to be too low to suppress the accumulation of type II collagen. On the other hand, the addition of EGCG markedly decreased both the matrix metalloproteinase-13 concentration in the supernatant of cell sheet culture and the type II collagen degradation activity in that supernatant. Taken together, EGCG may enhance the accumulation of type II collagen by suppressing type II collagen degradation. Addition of EGCG to the sheet culture of MSC markedly increased not only wet weight (A) and sGAG amount (B) but also type II collagen amount (C) in the cartilage-like cell sheet. [ABSTRACT FROM PUBLISHER]

Abstract: The effect of the addition of monosaccharides composing glycosaminoglycans (GAGs) on the accumulation of type 2 collagen (COL(II)) in a three-dimensional (3D) culture of porcine chondrocyte cells was investigated for possible application to cartilage regenerative medicine. Primary chondrocytes from porcine cartilage were cultivated in three-dimension employing atelo collagen gel for 3 weeks with the addition of several saccharides. The addition of d-glucuronic acid (d-GlcA), N-acetyl-d-galactosamine (d-GalNAc), chondroitin sulfate C (CSC), d-galactose, N-acetyl-d-glucosamine, and l-iduronic acid increased markedly not aggrecan but COL(II) accumulation although the addition of d-fructose and d-mannose not composing GAGs did not show such an effect. The addition of d-GlcA and d-GalNAc had no synergistic effect. The addition of CSC, d-GlcA, and d-GalNAc also increased COL(II) mRNA expression while aggrecan mRNA expression was not increased by these compounds. In conclusion, the addition of monosaccharides composing GAGs might be valuable for increasing COL(II) accumulation in the 3D culture of chondrocytes. [Copyright &y& Elsevier]

The effect of glycosaminoglycan addition on a three-dimensional (3D) culture of porcine chondrocyte cells was investigated with a view to use in cartilage regenerative medicine. Chondroitin sulfate C increased the mRNA expression of type 2 collagen, while chondroitin sulfate A did not. Hyaluronic acid of high molecular weight markedly decreased the mRNA expression of both aggrecan and type 2 collagen, although hyaluronic acid of low molecular weight showed no apparent effect. [Copyright &y& Elsevier]

Collagen gel and a copolymer mesh of polylactate and polyglucuronic acid (PLGA) were combined for a three-dimensional (3D) culture of chondrocyte cells having both uniform cell distribution and mechanical strength. Although the 3D culture in 96-multi-wells caused decreases in the glucose consumption rate and cell density in the latter stages of cultivation, transfer of the culture gel from a 96-multi-well plate to a 24-multi-well plate and an increase in medium volume effectively increased the glucose consumption rate and the accumulation of glycosaminoglycan (GAG) in the gel. The reason for the decrease in glucose consumption rate in a 96-multi-well plate was not the depletion of glucose or the accumulation of lactate in the gel, but the accumulation of degradation products of PLGA. [Copyright &y& Elsevier]

Abstract: Porcine articular cartilage tissue was crushed using Multi-Beads-Shocker™ and digested with trypsin and collagenase type II. Chondrocyte cell yield was maximized by automatic crushing for 10 s, which was 4 times that by manual dicing with a surgical blade and might be useful for the automation of the cell processing. [Copyright &y& Elsevier]

Noggin is a major extracellular antagonist to bone morphogenetic proteins (BMPs) which binds to BMPs and blocks binding of them to BMP-specific receptors and negatively regulates BMP-induced osteoblastic differentiation. In this study, we investigated the effect of noggin silencing by transfection of small interfering RNA (siRNA) on BMP-induced osteoblastic differentiation in vitro and ectopic bone formation in vivo induced by recombinant human BMP-2 (rhBMP-2). Noggin mRNA expression was up-regulated in response to rhBMP-2 in C2C12 cells, a myoblastic cell line, in dose- and time-dependent fashion as determined by real-time RT-PCR assay. Silencing of noggin expression by transfection of noggin siRNA suppressed BMP-stimulated noggin expression, resulting in acceleration of BMP-induced osteoblastic differentiation. For in vivo noggin silencing, siRNA was injected locally into back muscles and transfected into local cells by electroporation, where rhBMP-2-retaining (5 microg) collagen disks had been surgically placed. The implants were harvested at 2 weeks after surgery from experimental and control group mice and analyzed by radiological and histological methods. As a result, bone mineral content of ossicles ectopically induced by rhBMP-2 was significantly increased by silencing of noggin. Our findings suggest that silencing of noggin enhances the osteoblastic differentiation of BMP-responding cells in vitro and new bone formation induced by rhBMP-2 in vivo by eliminating negative regulation of the effects of BMP. RNA interference might be useful for intensifying the effects of BMP in promoting new bone (callus) formation in repair of damaged bone. [ABSTRACT FROM AUTHOR]

Abstract: To examine how fibroblast growth factor-2 (FGF-2) affects the BMP signaling pathway during bone morphogenetic protein-2 (BMP-2)-induced ectopic bone formation, we implanted type I collagen disks containing constant amounts of BMP-2 (5 μg) and varying amounts of FGF-2 onto the back muscles of adult male mice. We then performed histological analyses and histomorphometry, and measured bone mineral density and radiopaque area on the discs 1, 2, and 3 weeks after implantation. We also determined the expression profiles of several genes involved in bone formation and the BMP signaling pathway in the muscle that had been adjacent to the implanted disc and in muscle-derived primary culture cells that had similarly been treated with a constant concentration of BMP-2 and a varying concentration of FGF-2. In the presence of a constant amount of BMP-2, we confirmed that low doses of FGF-2 increased ectopic bone formation in vivo and high doses inhibited bone formation. Northern and/or Western blots of recovered muscle from the in vivo experiment and treated muscle-derived primary culture cells from the in vitro experiment revealed that low doses of FGF-2, but not high doses, increased the expression BMP receptor (BMPR)-1B, phosphorylated Smad1, Noggin, and Osteocalcin. Our results indicate that low-dose FGF-2 may facilitate BMP-2-induced ectopic bone formation by altering the expression of BMPRs on the surface of bone forming progenitor cells. They also indicate that the inhibitory effect of high-dose FGF-2 is not mediated via increased expression of the BMP inhibitor Noggin. [Copyright &y& Elsevier]