Methodology is described for the culture of avian and mammalian chondrocytes in ionotrophically gelled "semi-solid" and "hollow" alginate beads. Chondrocytes grown in "semi-solid" gels exhibited a spherical shape as opposed to a fibroblastic morphology observed in monolayer culture. In the "semi-solid" beads, the cells grew as small clumps and as large aggregates. The aggregates were round or elliptical in appearance and surrounded by a dense Alcian Blue positive halo. Preliminary studies with collagen and chitosan matrixes encapsulated in "hollow" beads suggest that cell growth and morphology are profoundly influenced by the composition of the cellular environment. Chondrocyte structure and function in the "semi-solid" and "hollow" beads were partially characterized by light microscopy, histochemical and biochemical means. The encapsulation methodology is readily applicable for the culture of chondrocytes in single beads, in multiwell dishes, or mass culture.

Hyaluronan synthesized in the epidermis has an exceptionally short half-life, indicative of its catabolism by epidermal keratinocytes. An intracellular pool of endogenously synthesized hyaluronan, from 1 to 20 fg/cell, inversely related to cell density, was observed in cultured rat epidermal keratinocytes. More than 80% of the intracellular hyaluronan was small (<90 kDa). Approximately 25% of newly synthesized hyaluronan was endocytosed by the keratinocytes and had a half-life of 2-3 h. A biotinylated aggrecan G 1 domain/link protein probe demonstrated hyaluronan in small vesicles of ϳ100 nm diameter close to the plasma membrane, and in large vesicles and multivesicular bodies up to 1300 nm diameter around the nucleus. Hyaluronan did not co-localize with markers of lysosomes. However, inhibition of lysosomal acidification with NH 4 Cl or chloroquine, or treating the cells with the hyaluronidase inhibitor apigenin increased intracellular hyaluronan staining, suggesting that it resided in prelysosomal endosomes. Competitive displacement of hyaluronan from surface receptors using hyaluronan decasaccharides, resulted in a rapid disappearance of this endosomal hyaluronan (t1 ⁄2 ϳ5 min), indicating its transitory nature. The ultrastructure of the hyaluronancontaining vesicles, co-localization with marker proteins for different vesicle types, and application of specific uptake inhibitors demonstrated that the formation of hyaluronan-containing vesicles did not involve clathrincoated pits or caveolae. Treatment of rat epidermal keratinocytes with the OX50 monoclonal antibody against the hyaluronan receptor CD44 increased endosomal hyaluronan. However, no CD44-hyaluronan co-localization was observed intracellularly unless endosomal trafficking was retarded by monensin, or cultivation at 20°C, suggesting CD44 recycling. Rat epidermal keratinocytes thus internalize a large proportion of their newly synthesized hyaluronan into non-clathrin-coated endosomes in a receptor mediated way, and rapidly transport it to slower degradation in the endosomal/lysosomal system.

Taste papillae are ectodermal specializations that serve to house and distribute the taste buds and their renewing cell populations in specific locations on the tongue. We previously showed that Sonic hedgehog (Shh) has a major role in regulating the number and spatial pattern of fungiform taste papillae on embryonic rat tongue, during a specific period of papilla formation from the prepapilla placode. Now we have immunolocalized the Shh protein and the Patched receptor protein (Ptc), and have tested potential roles for Shh in formation of the tongue, emergence of papilla placodes, development of papilla number and size, and maintenance of papillae after morphogenesis is advanced. Cultures of entire embryonic mandible or tongues from gestational days 12 to 18 [gestational or embryonic days (E)12-E18] were used, in which tongues and papillae develop with native spatial, temporal, and molecular characteristics. The Shh signaling pathway was disrupted with addition of cyclopamine, jervine, or the 5E1 blocking antibody. Shh and Ptc proteins are diffuse in prelingual tissue and early tongue swellings, and are progressively restricted to papilla placodes and then to regions of developing papillae. Ptc encircles the dense Shh immunoproduct in papillae at various stages. When the Shh signal is disrupted in cultures of E12 mandible, tongue formation is completely prevented. At later stages of tongue culture initiation, Shh signal disruption alters development of tongue shape (E13) and results in a repatterned fungiform papilla distribution that does not respect normally papilla-free tongue regions (E13-E14). Only a few hours of Shh signal disruption can irreversibly alter number and location of fungiform papillae on anterior tongue and elicit papilla formation on the intermolar eminence. However, once papillae are well formed (E16-E18), Shh apparently does not have a clear role in papilla maintenance, nor does the tongue retain competency to add fungiform papillae in atypical locations. Our data not only provide evidence for inductive and morphogenetic roles for Shh in tongue and fungiform papilla formation, but also suggest that Shh functions to maintain the interpapilla space and papilla-free lingual regions. We propose a model for Shh function at high concentration to form and maintain papillae and, at low concentration, to activate between-papilla genes that maintain a papilla-free epithelium.

From time of embryonic emergence, the gustatory papilla types on the mammalian tongue have stereotypic anterior and posterior tongue locations. Furthermore, on anterior tongue, the fungiform papillae are patterned in rows. Among the many molecules that have potential roles in regulating papilla location and pattern, Sonic hedgehog (Shh) has been localized within early tongue and developing papillae. We used an embryonic, tongue organ culture system that retains temporal, spatial, and molecular characteristics of in vivo taste papilla morphogenesis and patterning to study the role of Shh in taste papilla development. Tongues from gestational day 14 rat embryos, when papillae are just beginning to emerge on dorsal tongue, were maintained in organ culture for 2 days. The steroidal alkaloids, cyclopamine and jervine, that specifically disrupt the Shh signaling pathway, or a Shh-blocking antibody were added to the standard culture medium. Controls included tongues cultured in the standard medium alone, and with addition of solanidine, an alkaloid that resembles cyclopamine structurally but that does not disrupt Shh signaling. In cultures with cyclopamine, jervine, or blocking antibody, fungiform papilla numbers doubled on the dorsal tongue with a distribution that essentially eliminated inter-papilla regions, compared with tongues in standard medium or solanidine. In addition, fungiform papillae developed on posterior oral tongue, just in front of and beside the single circumvallate papilla, regions where fungiform papillae do not typically develop. The Shh protein was in all fungiform papillae in embryonic tongues, and tongue cultures with standard medium or cyclopamine, and was conspicuously localized in the basement membrane region of the papillae. Ptc protein had a similar distribution to Shh, although the immunoproduct was more diffuse. Fungiform papillae did not develop on pharyngeal or ventral tongue in cyclopamine and jervine cultures, or in the tongue midline furrow, nor was development of the single circumvallate papilla altered. The results demonstrate a prominent role for Shh in fungiform papilla induction and patterning and indicate differences in morphogenetic control of fungiform and circumvallate papilla development and numbers. Furthermore, a previously unknown, broad competence of dorsal lingual epithelium to form fungiform papillae on both anterior and posterior oral tongue is revealed.

Abundant hyaluronan is present between epidermal keratinocytes. However, virtually nothing is known regarding its organization in the limited extracellular space between these cells. We have used metabolic labeling with [ 3 H]glucosamine and [ 35 S]sulfate and a hyaluronan-specific biotinylated probe to study the metabolism of hyaluronan and its localization in monolayer cultures of a rat epidermal keratinocyte cell line. Hyaluronan (ϳ20 fg/cell) was present on the apical and lateral surfaces of the cells in two nearly equal pools, either in patches (ϳ160/cell) or diffusely spread. The hyaluronan in the patches is bound to CD44 as indicated by co-localization with an antibody to CD44, and by displacement with hyaluronan decasaccharides as well as with an antibody that blocks hyaluronan binding to CD44. The inability of hyaluronan oligomers shorter than 10 monosaccharides to displace hyaluronan suggests that CD44 dimerization or cooperative interactions are required for tight binding. The diffuse hyaluronan pool is likely bound to hyaluronan synthase during its biosynthesis.Hyaluronan is well known as a constituent of connective tissue extracellular matrices, but more recent studies have also demonstrated its abundance in stratified squamous epithelia including the epidermis (1-3). In contrast with connective tissue extracellular matrices that contain mixtures of collagens, fibronectins, other multiadhesive glycoproteins, proteoglycans, and hyaluronan, hyaluronan is the only known extracellular matrix macromolecule present in high concentration, ϳ2 mg/ ml, in the small extracellular space between adjacent epithelial cells (keratinocytes) that form the epidermis (4, 5). Additionally, studies of human skin organ cultures have shown that the hyaluronan within the epidermis is rapidly turned over (6), an observation that suggests that the epidermis possesses efficient mechanisms to catabolize hyaluronan that are closely coordinated with its synthesis.Although the coating of keratinocytes by hyaluronan is not generally appreciated, it is widely known that cell types of mesodermal origin (7), including fibroblasts (8), chondrocytes (9, 10), and mesothelial cells (11) display surface coats, often several micrometers in thickness, visualized indirectly as a domain excluding particles such as red blood cells. These coats 1) are removed by digestion with highly specific hyaluronidase, 2) can be stabilized by the serum-derived protein inter-␣-trypsin inhibitor which interacts with hyaluronan (12-14), and 3) can be increased in size and reinforced by proteoglycans that bind specifically to hyaluronan (15, 16).Extracellular hyaluronan is often anchored to CD44, a ubiquitous, abundant, and structurally variable plasma membrane receptor that has a hyaluronan binding domain (17). Smaller amounts of hyaluronan may bind to RHAMM, a receptor involved in cell motility and cell transformation through hyaluronan-dependent signaling involving tyrosine phosphorylation (18). In addition, some cell-surface hyaluronan appears to remain tet...