The quality of articular cartilage engineered using a cell-polymer construct depends, in part, on the chemical composition of the biomaterial and whether that biomaterial can support the chondrocytic phenotype. Acknowledging the supportive influence of tissue-specific matrix molecules on the chondrocytic phenotype, we have combined chondroitin sulfate-A (CSA) and chitosan, a glycosaminoglycan (GAG) analog, to develop a novel biomaterial to support chondrogenesis. Chitosan is a polycationic repeating monosaccharide of beta-1,4-linked glucosamine monomers with randomly located N-acetyl glucosamine units. Chitosan may be combined with the polyanionic CSA such that ionic crosslinking results in hydrogel formation. Bovine primary articular chondrocytes, when seeded onto a thin layer of CSA-chitosan, form discrete, focal adhesions to the material and maintain many characteristics of the differentiated chondrocytic phenotype, including round morphology, limited mitosis, collagen type II, and proteoglycan production. Our findings suggest CSA-chitosan may be well suited as a carrier material for the transplant of autologous chondrocytes or as a scaffold for the tissue engineering of cartilage-like tissue.

The addition of human recombinant interleukin-1beta (IL-1beta) to cultures of lapine articular chondrocytes provoked the synthesis of large amounts of NO and reduced the production of type-II collagen. NG-Monomethyl-l-arginine (L-NMA), an inhibitor of NO synthase, strongly suppressed the production of NO and partially relieved the inhibition of collagen synthesis in response to IL-1beta. The NO donor S-nitrosoacetylpenicillamine (SNAP), on the other hand, inhibited collagen production. IL-1 lowered the abundance of Col2A1 mRNA in an NO-independent manner. Collectively, these data indicate that IL-1 suppresses collagen synthesis at two levels: a pretranslational level which is NO-independent, and a translational or post-translational level which is NO-mediated. These effects are presumably specific as L-NMA and SNAP had no effect on total protein synthesis or on the distribution of newly synthesized proteins between the cellular and extracellular compartments. Prolyl hydroxylase is an important enzyme in the post-translational processing of collagen, and its regulation and cofactor requirements suggest possible sensitivity to NO. Extracts of cells treated with IL-1 or SNAP had lower prolyl hydroxylase activity, and L-NMA was partially able to reverse the effects of IL-1. These data suggest that prolyl hydroxylase might indeed be a target for NO. Because underhydroxylated collagen monomers fail to anneal into stable triple helices, they are degraded intracellularly. Inhibition of prolyl hydroxylase by NO might thus account for the suppressive effect of this radical on collagen synthesis.

The full-length cDNA of mouse K12 keratin was characterized by sequencing overlapping cDNA clones isolated from a mouse cornea cDNA library. Using Northern blot hybridization, the radio-labeled cDNA hybridized to a 1.9 kb mRNA from adult cornea, but not from other mouse tissues including snout, esophagus, tongue, and skin. During mouse development, corneas do not express K12 mRNA until 4 days postnatal when the epithelium begins to stratify as judged by Northern blot and in situ hybridization. In situ hybridization with 3H-labeled cDNA probe and immunohistochemical studies with antibodies against a synthetic oligo-peptide deduced from rabbit K12 cDNA demonstrate that this mouse K12 keratin is expressed in all cell layers of adult corneal epithelium, and the suprabasal layers, but not the basal layer of the limbal epithelium. Epidermal growth factor (EGF) has been shown to promote epithelium stratification of cultured chicken and human corneas in vitro. To examine whether EGF can promote K12 expression, EGF was administered to neonatal mice. The results indicate that EGF retards K12 expression by corneal epithelial cells, even though it promotes corneal epithelial stratification during mouse development. Taken together, our results demonstrate that the expression of K12 keratin is cornea-specific, differentiation-dependent, and developmentally regulated.

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