Endochondral skeletal development involves the condensation of mesenchymal cells, their differentiation into chondrocytes, followed by chondrocyte maturation, hypertrophy, and matrix mineralization, and replacement by osteoblasts. The Wnt family of secreted proteins have been shown to play important roles in vertebrate limb formation. To examine the role(s) of Wnt members and their transmembrane-spanning receptor(s), Frizzled (fz), we retrovirally misexpressed Wnt-5a, Wnt-7a, chicken frizzled-1 (Chfz-1), and frizzled-7 (Chfz-7) in long-term (21 day) high density, micromass cultures of stage 23/24 chick embryonic limb mesenchyme. This culture system recapitulates in vitro the entire differentiation (days 1-10), growth (days 5-12), and maturation and hypertrophy (from day 12 on) program of cartilage development. Wnt-7a misexpression severely inhibited chondrogenesis from day 7 onward. Wnt-5a misexpression resulted in a poor hypertrophic phenotype by day 14. Chfz-7 misexpression caused a slight delay of chondrocyte maturation based on histology, whereas Chfz-1 misexpression did not affect the chondrogenic phenotype. Misexpression of all Wnt members decreased collagen type X expression and alkaline phosphatase activity at day 21. Our findings implicate functional role(s) for Wnt signaling throughout embryonic cartilage development, and show the utility of the long-term in vitro limb mesenchyme culture system for such studies.

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Products of the Frizzled family of tissue polarity genes have been identified as putative receptors for the Wnt family of signaling molecules. Wnt-signaling is implicated in the regulation of limb mesenchymal chondrogenesis, and our recent study indicates that N-cadherin and related activities are functionally involved in Wnt-7a-mediated inhibition of chondrogenesis. By using an in vitro high-density micromass culture system of chick limb mesenchymal cells, we have analyzed the spatiotemporal expression patterns and the effects on chondrogenesis of RCAS retroviral-mediated misexpression of Chfz-1 and Chfz-7, two Frizzled genes implicated in chondrogenic regulation. Chfz-1 expression was localized at areas surrounding the cartilaginous nodules at all time points examined, whereas Chfz-7 expression was limited to cellular aggregates during initial mesenchymal condensation, and subsequently was down-regulated from the centers toward the periphery of cartilage nodules at the time of chondrogenic differentiation, resembling the pattern of N-cadherin expression. Chondrogenesis in vitro was inhibited and limited to a smaller area of the culture upon misexpression of Chfz-7, but not affected by Chfz-1 misexpression. Analyses of cellular condensation and chondrogenic differentiation showed that the inhibitory action of Chfz-7 is unlikely to be at the chondrogenic differentiation step, but instead affects the earlier precartilage aggregate formation event. At 24 hr, expression of N-cadherin, a key component of the cellular condensation phase of chondrogenesis, was delayed/ suppressed in Chfz-7 misexpressing cultures, and was limited to a significantly smaller cellular condensation area within the entire culture at 48 hr, when compared with control cultures. Chfz-1 misexpressing cultures appeared similar to control cultures at all time points. However, neither Chfz-1 nor Chfz-7 misexpression affected mesenchymal cell proliferation in vitro. These results suggest that Chfz-7 is active in regulating N-cadherin expression during the process of limb mesenchymal chondrogenesis and that Chfz-1 and Chfz-7 are involved in different Wnt-signaling pathways.

Reactive oxygen species produced by activated neutrophils and monocytes are thought to be involved in mediating the loss of collagen and other matrix proteins at sites of inflammation. To evaluate their potential to oxidize the pyridinoline (Pyd) cross-links found in collagen types I and II, we reacted hydrogen peroxide (H 2 O 2 ), hypochlorous acid/hypochlorite (HOCl/OCl ؊ ), and singlet oxygen (O 2 ( 1 ⌬g)) with the Pyd substitutes, pyridoxamine dihydrochloride and vitamin B 6 , which share the same chemical structure and spectral properties of Pyd cross-links. Neither H 2 O 2 (125-500 M) nor O 2 ( 1 ⌬g) (10 -25 M) significantly changed the spectral properties of pyridoxamine or vitamin B 6 . Reaction of HOCl/OCl ؊ (12.5-50 M) with pyridoxamine at pH 7.2 resulted in a concentration-dependent appearance of two new absorbance peaks and a decrease in fluorescence at 400 nm (excitation 325 nm). The new absorbance peaks correlated with the formation of an N-chloramine and the product of its subsequent reaction with pyridoxamine. In contrast, the extent to which HOCl reacted with vitamin B 6 , which lacks a primary amine group, was variable at this pH. At lysosomal pH 5.5, Cl 2 /HOCl/OCl ؊ reacted with both pyridoxamine and vitamin B 6 . Four of the chlorinated products of this reaction were identified by gas chromatography-mass spectrometry and included 3-chloropyridinium, an aldehyde, and several chlorinated products with disrupted rings. To evaluate the effects of Cl 2 /HOCl/OCl ؊ on Pyd cross-links in collagen, we exposed bone collagen type I and articular cartilage type II to HOCl. Treatment of either collagen type with HOCl at pH 5.0 or 7.2 resulted in the oxidation of amine groups and, for collagen type II, the specific decrease in Pyd cross-link fluorescence, suggesting that during inflammation both oxidations may be used by neutrophils and monocytes to promote the loss of matrix integrity.Inflammation associated with articular cartilage, bone, and dentin surfaces is characterized by accumulation, adhesion, and activation of neutrophils and monocytes, which results in the destruction of cartilage and the loss of bone at these sites (1-4). These effects are thought to be mediated in part by the production of reactive oxygen species (ROS), 1 a group of reactants that includes superoxide (O 2 . ), hydrogen peroxide (H 2 O 2 ), and the highly reactive species hypochlorous acid (HOCl) (5-8).ROS are produced by neutrophils and monocytes after they are recruited from the circulation to extravascular spaces. Once outside of the circulation, they adhere to extracellular matrix proteins (ECM) and undergo activation, which results in the production of ROS and release of proteolytic enzymes directly onto the matrix surface (4, 9 -13). In inflammation associated with arthritic joints, the accumulation and activation of neutrophils and monocytes and increased synovial cell formation result in the loss of synovial membrane integrity and eventually to irreversible damage and destruction of articular cartilage in the affli...