Combining stretching and gallic acid to decrease inflammation indices and promote extracellular matrix production in osteoarthritic human articular chondrocytes.

Osteoarthritis (OA) patients undergo cartilage degradation and experience painful joint swelling. OA symptoms are caused by inflammatory molecules and the upregulation of catabolic genes leading to the breakdown of cartilage extracellular matrix (ECM). Here, we investigate the effects of gallic acid (GA) and mechanical stretching on the expression of anabolic and catabolic genes and restoring ECM production by osteoarthritic human articular chondrocytes (hAChs) cultured in monolayers. hAChs were seeded onto conventional plates or silicone chambers with or without 100 µM GA. A 5% cyclic tensile strain (CTS) was applied to the silicone chambers and the deposition of collagen and glycosaminoglycan, and gene expressions of collagen types II (COL2A1), XI (COL11A2), I (COL1A1), and X (COL10A1), and matrix metalloproteinases (MMP-1 and MMP-13) as inflammation markers, were quantified. CTS and GA acted synergistically to promote the deposition of collagen and glycosaminoglycan in the ECM by 14- and 7-fold, respectively. Furthermore, the synergistic stimuli selectively upregulated the expression of cartilage-specific proteins, COL11A2 by 7-fold, and COL2A1 by 47-fold, and, in contrast, downregulated the expression of MMP-1 by 2.5-fold and MMP-13 by 125-fold. GA supplementation with CTS is a promising approach for restoring osteoarthritic hAChs ECM production ability making them suitable for complex tissue engineering applications.

Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells (MSCs) in different hydrogels: influence of collagen type II extracellular matrix on MSC chondrogenesis.

Bone marrow mesenchymal stem cells (MSCs) are candidate cells for cartilage tissue engineering. This is due to their ability to undergo chondrogenic differentiation after extensive expansion in vitro and stimulation with various biomaterials in three-dimensional (3-D) systems. Collagen type II is one of the major components of the hyaline cartilage and plays a key role in maintaining chondrocyte function. This study aimed at analyzing the MSC chondrogenic response during culture in different types of extracellular matrix (ECM) with a focus on the influence of collagen type II on MSC chondrogenesis. Bovine MSCs were cultured in monolayer as well as in alginate and collagen type I and II hydrogels, in both serum free medium and medium supplemented with transforming growth factor (TGF) beta1. Chondrogenic differentiation was detected after 3 days of culture in 3-D hydrogels, by examining the presence of glycosaminoglycan and newly synthesized collagen type II in the ECM. Differentiation was most prominent in cells cultured in collagen type II hydrogel, and it increased in a time-dependent manner. The expression levels of the of chondrocyte specific genes: sox9, collagen type II, aggrecan, and COMP were measured by quantitative "Real Time" RT-PCR, and genes distribution in the hydrogel beads were localized by in situ hybridization. All genes were upregulated by the presence of collagen, particularly type II, in the ECM. Additionally, the chondrogenic influence of TGF beta1 on MSCs cultured in collagen-incorporated ECM was analyzed. TGF beta1 and dexamethasone treatment in the presence of collagen type II provided more favorable conditions for expression of the chondrogenic phenotype. In this study, we demonstrated that collagen type II alone has the potential to induce and maintain MSC chondrogenesis, and prior interaction with TGF beta1 to enhance the differentiation.

Gene expression of connective tissue growth factor (CTGF/CCN2) in calcifying tissues of normal and cbfa1-null mutant mice in late stage of embryonic development.

Connective tissue growth factor (CTGF/CCN2), one of the most recently described growth factors, is produced by chondrocytes, vascular endothelial cells, and transforming growth factor (TGF)-beta-stimulated fibroblasts. CTGF was isolated from a chondrosarcoma-derived chondrocytic cell line, HCS-2/8, and found to be normally expressed in cartilage tissues, especially in hypertrophic chondrocytes, and also to stimulate both the proliferation and the differentiation of chondrocytes in vitro. Therefore, CTGF is thought to be one of the most important regulators of endochondral ossification in vivo. Herein we describe the expression pattern of the ctgf gene in the calcifying tissues of normal developing mouse embryos in comparison with that in core binding factor a1 (Cbfa1)-targeted mutant (cbfa1-null) mouse embryos, in which impaired development and growth were characteristically observed in the skeletal system. After 15 days of development (E15), the expression of ctgf was detected in the zone of hypertrophy and provisional calcification, in which ossification proceeds toward the epiphysis during the skeletal development of the mouse embryo. Furthermore, ctgf was expressed in developing molar and incisal tooth germs around the perinatal stage. However, no expression of the gene was found in the cbfa1-null mouse embryos. These results indicate that CTGF may have certain important roles in the development of the calcifying tissues in the mouse embryo.

Rac1/Cdc42 and RhoA GTPases antagonistically regulate chondrocyte proliferation, hypertrophy, and apoptosis.

UNLABELLED: The intracellular signaling pathways controlling chondrocyte physiology are largely unknown. Here we show that the small GTPases, Rac1 and Cdc42, accelerate the rate of chondrocyte differentiation and apoptosis, thereby antagonizing the activity of RhoA. These results identify Rac1 and Cdc42 pathways as novel regulators of cartilage development. INTRODUCTION: Proliferation, hypertrophic differentiation, and ultimate apoptosis of chondrocytes regulate endochondral bone growth and development, but the intracellular signaling pathways controlling chondrocyte biology are incompletely understood. In this study, we investigated the role of the small GTPases Rac1 and Cdc42 in chondrocytes. MATERIALS AND METHODS: Rac1 and Cdc42 expression during chondrogenic differentiation was assessed by RT-PCR and Western blotting. Effects of Rac1 and Cdc42 on parameters of chondrocyte biology were studied using transient transfections into primary mouse chondrocytes and stable transfections of the chondrogenic cell line ATDC5. Luciferase assays, RT-PCR, cell proliferation, alkaline phosphatases assays, staining procedures, TUNEL assays, and caspase activity assays were performed to study the chondrocyte response to overexpression of Rac1 and Cdc42 proteins. Activation of the p38 pathway was analyzed using Western blotting with phospho-specific antibodies, and mitogen-activated protein (MAP) kinase pathways were inhibited using pharmacological approaches. RESULTS AND CONCLUSIONS: Rac1 and Cdc42 activities are required for maximal activity of the collagen X promoter, a hypertrophic marker, in primary chondrocytes, suggesting essential roles of these GTPases in chondrocyte hypertrophy. Overexpression of Rac1 or Cdc42 in chondrogenic ATDC5 cells results in reductions in cell numbers and marked acceleration of hypertrophic differentiation, thus opposing the effects of the related GTPase RhoA. Rac1 and Cdc42 also induce accelerated chondrocyte apoptosis, as shown by TUNEL and caspase activity assays and changes in cell morphology and actin organization. Rac1 and Cdc42 overexpression results in activation of the p38 MAP kinase pathway in ATDC5 cells, and pharmacological inhibition of p38 signaling blocks the effects of Rac1 and Cdc42 overexpression on hypertrophy and apoptosis. Our results therefore suggest that Rac1 and Cdc42 signaling accelerates progression through the chondrocyte life cycle in a p38-dependent fashion and antagonizes RhoA signaling pathways in chondrocyte proliferation, hypertrophy, and apoptosis.

Hand involvement in Schmid metaphyseal chondrodysplasia.

Schmid metaphyseal chondrodysplasia (Schmid MCD, MIM 156500) is caused by mutations in the COL10A1 gene and is clinically characterized by short stature, bowed legs, and a waddling gait. Radiographic findings include anterior cupping, sclerosis and splaying of the ribs, diffuse metaphyseal flaring, and irregularity that is most pronounced at the knees, coxa vara, and femoral bowing. We reviewed the radiographs of Schmid MCD patients at the International Skeletal Dysplasia Registry in Los Angeles for evidence of hand involvement. We found hand involvement in 47% (7/15) of cases included in our analysis. These changes were subtle and consisted of shortening of the tubular bones and metaphyseal cupping of the proximal phalanges and metacarpals. Mild hand involvement is a common feature of Schmid MCD.

Retinoic acid stimulates chondrocyte differentiation and enhances bone morphogenetic protein effects through induction of Smad1 and Smad5.

Whereas bone morphogenetic protein (BMP)-signaling events induce maturational characteristics in vitro, recent evidence suggests that the effects of other regulators might be mediated through BMP-signaling events. The present study examines the mechanism through which retinoic acid (RA) stimulates differentiation in chicken embryonic caudal sternal chondrocyte cultures. Both RA and BMP-2 induced expression of the chondrocyte maturational marker, colX, in chondrocyte cultures by 8 d. Though the RA effect was small, it synergistically enhanced the effect of BMP-2 on colX and phosphatase activity. Inhibition of either RA or BMP signaling, with selective inhibitors, interfered with the inductive effects of these agents but also inhibited the complementary pathway, demonstrating a codependence of RA and BMP signaling during chondrocyte maturation. BMP-2 did not enhance the effects of RA on an RA-responsive reporter construct, but RA enhanced basal activity and synergistically enhanced BMP-2 stimulation of the BMP-responsive chicken type X collagen reporter. A similar synergistic interaction between RA and BMP-2 was observed on colX expression. RA did not increase the expression of the type IA BMP receptor but did markedly up-regulate the expression of Smad1 and Smad5 proteins, important participants in the BMP pathway. Inhibition of RA signaling, with the selective inhibitor AGN 193109, blocked RA-mediated induction of the Smad proteins and chondrocyte differentiation. These findings demonstrate that RA induces the expression of BMP-signaling molecules and enhances BMP effects in chondrocytes.