Thyroid hormone, insulin, and glucocorticoids are sufficient to support chondrocyte differentiation to hypertrophy: a serum-free analysis.

Chondrocytes from chicken embryo tibia can be maintained in culture as adherent cells in Coon's modified Ham's F-12 medium supplemented with 10% FCS. In this condition, they dedifferentiate, losing type II collagen expression in favor of type I collagen synthesis. Their differentiation to hypertrophy can be obtained by transferring them to suspension culture.

The control of polarized integrin topography and the organization of adhesion-related cytoskeleton in normal human keratinocytes depend upon number of passages in culture and ionic environment.

Keratinocyte adhesion to basal lamina and lateral interactions among basal epidermal cells are mediated, besides other molecules, by integrin receptors that are sorted to defined membrane domains. The hemidesmosome-associated integrin alpha 6 beta 4 is sharply localized to the basal surface of basal cells while alpha 2 beta 1 and alpha 3 beta 1 are enriched laterally. This integrin sorting pattern is perfectly reproducible in vitro by cultured keratinocytes and takes place progressively in primary or secondary culture in the presence of 1.

Expression of epidermal growth factor receptor and transforming growth factor alpha in human larynx carcinoma.

Altered expression of growth factors and growth factor receptors is frequently described in human tumors and human tumor cell lines. This further supports the hypothesis that oncogenesis is due to the subversion of mitogen-responsive pathways. The aim of this study was to investigate the expression of epidermal growth factor receptor (EGFR) and transforming growth factor alpha (TGF alpha) in 13 larynx carcinomas and 2 carcinomas of the oral cavity.

In vitro differentiation of mouse embryo chondrocytes: requirement for ascorbic acid.

Chondrocytes enzymatically dissociated from 13-day-old mouse embryo tibia grow in monolayer culture with a fibroblast-like phenotype and express high levels of type I collagen. Chondrogenesis can be induced by transferring the adherent cells in suspension culture and maintaining them in the constant presence of mouse embryo extract. Round shaping of the cells and formation of multicellular aggregates rapidly follow the passage in anchorage-independent conditions.

Expression, topography, and function of integrin receptors are severely altered in keratinocytes from involved and uninvolved psoriatic skin.

Psoriasis is a hyperproliferative cutaneous disease of unknown etiology and etiopathogenesis. Alteration of keratinocyte adhesiveness to basal lamina has been proposed as the initial disturbance leading to poorly controlled proliferation. Keratinocyte adhesion to basal lamina and lateral interactions among basal epidermal cells are mediated, besides other molecules, by integrin receptors that are segregated to discrete membrane domains.

Treatment of leg ulcers with cryopreserved allogeneic cultured epithelium. A multicenter study.

Background: In the past few years, several authors have described the usefulness of cultured allogeneic epidermal sheets in promoting wound healing of burns, leg ulcers, and donor sites. This study reports clinical results obtained by different departments in the treatment of chronic leg ulcers by cryopreserved cultured allogeneic epithelium. The freezing procedure and the assessment of viability of the cryopreserved epithelium are also described.

Cell condensation in chondrogenic differentiation.

Reduction of intercellular spaces in the areas of prospective cartilage and bone formation (precartilage condensation) precedes chondrogenesis and may represent an important step in the process of cartilage differentiation during limb skeletogenesis. We have attempted to clarify the role of the microenvironment established during cell condensation, taking advantage of a tissue culture model system that allows condensation (i.e.

In vitro paracrine regulation of human keratinocyte growth by fibroblast-derived insulin-like growth factors.

Human keratinocytes isolated from a skin biopsy and cultured in vitro on a feeder-layer of irradiated fibroblasts reconstitute a stratified squamous epithelium suitable for grafting onto patients suffering from large burn wounds. Since conditioned medium from 3T3-J2 cells can partially substitute for the intact feeder-layer, we studied the possible involvement of insulin-like growth factors acting in a paracrine fashion. IGFs were measured (after Sephadex G-50 gel-chromatography in acid conditions) in media conditioned by a feeder-layer of lethally irradiated 3T3-J2 fibroblasts on which keratinocytes were grown.

Heat-shock response in cultured chick embryo chondrocytes. Osteonectin is a secreted heat-shock protein.

We investigated the induction of specific protein expression by heat shock in dedifferentiated and hypertrophic chick embryo chondrocytes in a culture system that allows 'in vitro' differentiation of cartilage cells [Castagnola, P., Moro, G., Descalzi-Cancedda, F.

Hypertrophic chondrocytes undergo further differentiation in culture.

Conditions have been defined for promoting growth and differentiation of hypertrophic chondrocytes obtained in culture starting from chick embryo tibiae. Hypertrophic chondrocytes, grown in suspension culture as described (Castagnola P., G.

Chondrogenic differentiation in chick embryo osteoblast cultures.

Expression of specific differentiation markers was investigated by histochemistry, immunofluorescence, and biosynthetic studies in osteoblasts outgrown from chips derived from tibia diaphyses of 18-day-old chick embryos. The starting osteoblast population expressed type I collagen and alkaline phosphatase in addition to other bone and cartilage markers as the lipocalin Ch21; the extracellular matrix deposited by these cells was not stainable for cartilage proteoglycans, and mineralization was observed when the culture was maintained in the presence of ascorbic acid, calcium and beta-glycerophosphate. During culture, clones of cells presenting a polygonal chondrocyte morphology and surrounded by an Alcian-positive matrix appeared in the cell population.

"HepG2/erythroid/brain" type glucose transporter (GLUT1) is highly expressed in human epidermis: keratinocyte differentiation affects GLUT1 levels in reconstituted epidermis.

In mature animals, the "HepG2/erythroid/brain" glucose transporter isoform (GLUT1) appears to be expressed at the highest levels at blood tissue barriers; however, these levels may still be lower than the levels of expression seen in fetal tissues. Also, glucose transporters might serve as water channels. Therefore, we decided to investigate GLUT1 expression in human epidermis, a very active tissue, in terms of metabolism, even if not directly vascularized.

Expression, regulation, and tissue distribution of the Ch21 protein during chicken embryogenesis.

The Ch21 protein is one of the marker proteins whose synthesis and secretion by differentiating tibia chondrocytes is progressively increased during chicken embryogenesis (Descalzi-Cancedda, F., Manduca, P., Tacchetti, C.

Constitutive myc expression impairs hypertrophy and calcification in cartilage.

The myc oncogene is expressed by proliferating quail embryo chondrocytes (QEC) grown as adherent cells and is repressed in QEC maintained in suspension culture. To investigate the interference of myc expression during chondrocyte differentiation, QEC were infected with a retrovirus carrying the v-myc oncogene (QEC-v-myc). Uninfected or helper virus-infected QEC were used as control.

In vitro effects of beta-carotene on human oral keratinocytes from precancerous lesions and squamous carcinoma.

Human keratinocytes, obtained from bioptic specimens of healthy and preneoplastic oral mucosa, and from human cell lines from oral cavity tumors (KB and SCC-25) were treated with beta-carotene (10 microM). The colony forming efficiency (CFE), the proliferation rate and the frequency of micronucleated cells were measured in these cultures. CFE was significantly reduced (p less than 0.

Growth-regulated synthesis and secretion of biologically active nerve growth factor by human keratinocytes.

Nerve growth factor (NGF) transcripts were identified in normal human keratinocytes in primary and secondary culture. The expression of the NGF mRNA was strongly down-regulated by corticosteroids and was maximal when keratinocytes were in the exponential phase of growth. Immunofluorescence studies on growing keratinocytes colonies and on elutriated keratinocytes obtained from growing colonies and mature stratified epithelium showed specific staining of the Golgi apparatus only in basal keratinocytes in the exponential phase of growth.

High expression levels of the "erythroid/brain" type glucose transporter (GLUT1) in the basal cells of human eye conjunctiva and oral mucosa reconstituted in culture.

The expression of the "erythroid/brain" type glucose transporter (GLUT1) seems to be a feature of "barrier" tissues, at least in humans. Recently, we reported that GLUT1 is highly expressed in the basal layers of either "authentic" human epidermis or human epidermis reconstituted in culture and that its expression seems to be related to keratinocyte differentiation. In this paper we demonstrate that GLUT1 is selectively expressed in the basal layers of either eye conjunctiva epithelia or oral mucosa, reconstituted in culture starting from 1-2 mm2 bioptic specimens of normal human tissue.

cDNA cloning and gene expression of chicken osteopontin. Expression of osteopontin mRNA in chondrocytes is enhanced by trypsin treatment of cells.

A cDNA clone, pCP15, specific for the chicken 66-kDa major bone phosphoprotein (osteopontin), was isolated from a subtracted library enriched in DNAs coding for mRNAs expressed in chicken differentiating chondrocytes. Northern blot analysis of RNAs extracted from several chick embryo tissues and organs, confirm and extend the observation that osteopontin mRNA expression is not restricted to tissues involved in phosphate metabolism. Osteopontin mRNA was detected in sternal resting chondrocytes at higher levels than in hypertrophic chondrocytes; therefore osteopontin gene transcription occurs in chondrocytes at many stages of differentiation.

Expression of anchorin CII mRNA by cultured chondrocytes.

The establishment of a cell culture system promoting chondrocyte differentiation has been utilized to better characterize phenotypic stages of chondrogenesis at the cellular level. Although the expression of the type II collagen gene has been studied during "in vitro" chondrocyte differentiation, little is known about the expression of the gene coding for its receptor: anchorin CII. The modulation of the anchorin mRNA steady state level in chick embryo chondrocytes at different developmental stages is described here.

Polarized expression of integrin receptors (alpha 6 beta 4, alpha 2 beta 1, alpha 3 beta 1, and alpha v beta 5) and their relationship with the cytoskeleton and basement membrane matrix in cultured human keratinocytes.

In human keratinocytes cultured in conditions which allow differentiation and stratification and are suitable to reconstitute a fully functional epidermis, alpha 6 beta 4 and two members of the beta 1 integrin family (alpha 2 beta 1 and alpha 3 beta 1) were respectively polarized to the basal and lateral domains of the plasmamembrane both in growing colonies and in the reconstituted epidermis. Conversely, the alpha v integrin subunit, presumably in association with beta 5, was expressed at the basal surface in growing and migrating but not in stationary keratinocytes. The integrin alpha 6 beta 4: (a) was organized in typical patches which often showed a "leopard skin" pattern where spots corresponded to microfilament-free areas; (b) was not associated with focal contacts containing vinculin and talin but rather corresponded to relatively removed contact areas of the basal membrane as shown by interference reflection microscopy; and (c) was coherent to patches of laminin secreted and deposited underneath the ventral membrane of individual cells.

Structural and functional studies of integrin receptors in cultured human keratinocytes.

In human keratinocytes cultured in conditions which allow differentiation and stratification and which are suitable to reconstitute a fully functional epidermis, the integrin alpha 6 beta 4 and two members of the beta 1 integrin family (alpha 2 beta 1 and alpha 3 beta 1) were polarized to the basal and lateral domains of the plasma membrane both in growing colonies and in the reconstituted epidermis. Conversely, alpha v beta 5 integrin was expressed at the basal surface in growing and migrating but not in stationary keratinocytes. The integrin alpha 6 beta 4 was organized in patches and spots corresponding to F-actin-free submembraneous areas and did not colocalize to focal contacts; moreover, alpha 6 beta 4 colocalized with patches of laminin deposited underneath the ventral membrane of individual cells.

The Ch21 protein, developmentally regulated in chick embryo, belongs to the superfamily of lipophilic molecule carrier proteins.

Ch21, a developmentally regulated low molecular weight protein observed in chick embryo skeletal tissues, is expressed "in vitro" by differentiating chondrocytes at a late stage of development. Here we report the complete amino acid sequence of the protein. 86% of the total amino acid sequence was deduced by sequences of 17 high performance liquid chromatography-separated proteolytic fragments and 33 amino acid residues at the amino-terminal end of protein purified from spent culture medium of hypertrophic chondrocytes.

Evidence that human oral epithelium reconstituted in vitro and transplanted onto patients with defects in the oral mucosa retains properties of the original donor site.

Normal human skin--derived keratinocytes cultured in vitro reconstitute a stratified epidermis suitable for grafting onto burn patients and patients with skin defects such as giant nevi or chronic leg ulcers. In vitro experiments and long-term studies of patients receiving cultured epidermis autografts on muscular fascia suggest that skin keratinocytes possess an intrinsic site specific differentiation program that is fully expressed only when the reconstituted epidermis is transplanted in vivo to different body sites. In this study we cultivated for the first time palate-derived epithelial cells that were able to reconstitute a palatal epithelium.