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Biomedical subjects

R Cancedda

Publications and source records attributed to R Cancedda.

At least 91 records · Page 5Linked to original sources
PubMed1992

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. A total of 30 ulcers were treated using 138 cryopreserved allografts. OBSERVATIONS: Twenty ulcers (66.6%) healed completely within 12 weeks. Four ulcers showed a 30% to 84.4% reduction in size by 3 weeks but did not heal completely; the remaining six ulcers did not show any improvement. A strong stimulation of granulation tissue formation and of reepithelialization from the wound edge were observed. RESULTS: The results indicate that frozen cultured epidermis, stored in a skin bank, is a valid and generally applicable alternative therapy for the treatment of chronic ulcers.

Aged↗
PubMed1991

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. The keratinocyte-derived NGF was secreted in a biologically active form as assessed by neurite induction in sensory neurons obtained from chick embryo dorsal root ganglia. Based on these data we suggest that the basal keratinocyte is the cell synthesizing and secreting NGF in the human adult epidermis. The paracrine secretion of NGF by keratinocytes might have a major role in regulating innervation, lymphocyte function, and melanocyte growth and differentiation in epidermal morphogenesis as well as during wound healing.

Animals↗
PubMed1991

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. The steady state level of osteopontin mRNA was enhanced by trypsin treatment of cultured cells. An increased level of osteopontin mRNA in quail chondrocytes constitutively expressing v-myc oncogene is also shown.

Amino Acid Sequence↗
PubMed1991

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. GLUT1 mRNA and protein levels are very high in conjunctiva and oral mucosa, 2-3 times higher than in epidermis reconstituted in culture. Taking into account its localization at the border of tissues not directly vascularized, but metabolically active, GLUT1 could play an important role in controlling the entry of glucose into these firmly guarded tissues.

Blotting, Northern↗
PubMed1991

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. The two beta 1 integrins (alpha 2 beta 1 and alpha 3 beta 1), both endowed with laminin receptor properties, were not associated with focal adhesions under experimental conditions allowing full epidermal maturation but matched the lateral position of vinculin (but not talin), cingulin, and desmoplakin, all makers of intercellular junctions. Often thin strips of laminin were observed in between the lateral aspects of individual basal keratinocytes. The integrin complex alpha v beta 5 had a topography similar to that of talin- and vinculin-containing focal adhesions mostly in the peripheral cells of expanding keratinocyte colonies and in coincidence with fibronectin strands. The discrete topography of beta 1 and beta 4 integrins has a functional role in the maintenance of the state of aggregation of cultured keratinocytes since lateral aggregation was impaired by antibodies to beta 1 whereas antibodies to beta 4 prevented cell-matrix adhesion (De Luca, M., R. N. Tamura, S. Kajiji, S. Bondanza, P. Rossino, R. Cancedda, P. C. Marchisio, and V. Quaranta. Proc. Natl. Acad. Sci. USA. 87:6888-6892). Moreover, the surface polarization of integrins followed attachment and depended both on the presence of Ca2+ in the medium and on the integrity of the cytoskeleton. We conclude that our in vitro functional tests and structural data suggest a correlation between the pattern of integrin expression on defined plasmamembrane domains and the mechanism of epidermal assembly.

Basement Membrane↗
PubMed1991

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 0.05) by beta-carotene treatment in cells from healthy mucosa and in KB cells. Decreases (p greater than 0.05; NS) were also observed in cells from pathological mucosa and in SCC-25 cells. Cell proliferation rate was not substantially affected by beta-carotene in all cultures. Finally, a decreased frequency of micronucleated cells was found in treated cultures, but significant reductions (p less than 0.05) were only observed in cultures from oral mucosa (healthy and pathological) as well as in KB cell cultures. Our results indicate that beta-carotene is able to reduce the clonogenic activity (CFE), even if it does not seem to influence cell proliferation, and that it has a protective effect against genotoxic damage.

Adolescent↗
PubMed1990

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 two beta 1 laminin receptor integrins (alpha 2 beta 1 and alpha 3 beta 1) were never found in the basal domain but matched the lateral position of vinculin (but not talin), cingulin and desmoplakins. Only the integrin complex alpha v beta 5 was associated with talin- and vinculin-containing focal adhesions mostly in the peripheral cells of expanding keratinocyte colonies and in coincidence with fibronectin strands. The topography of beta 1 and beta 4 integrins reflects a functional role in adhesion and in the maintenance of the state of aggregation of cultured keratinocytes since lateral aggregation was impaired by antibodies to beta 1 whereas antibodies to beta 4 prevented cell-matrix adhesion.

Calcium↗
PubMed1990

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. Furthermore we isolated by molecular cloning the corresponding cDNA and determined its nucleotide sequence. By combining protein and nucleotide sequence data we determined the primary structure of the entire Ch21. It consists of 158 amino acids and has a molecular mass of 18.065 kDa. Computer-assisted analysis showed that the Ch21 belongs to the superfamily of low molecular weight proteins sharing a basic framework for binding and transport of small hydrophobic molecules.

Amino Acid Sequence↗
PubMed1990

The amino terminal sequence of the developmentally regulated Ch21 protein shows homology with amino terminal sequences of low molecular weight proteins binding hydrophobic molecules.

Ch21 protein, a developmentally regulated chick embryo protein of 21,000 apparent molecular weight, was purified from culture medium of hypertrophic chondrocytes. The purification method included a DEAE cellulose chromatography column, a CM cellulose chromatography column and a HPLC molecular sieve column. The amino acid sequence of the amino terminal end of the protein was determined. Computer assisted analysis showed significant homology between this sequence and the amino terminal sequences of proteins that belong to the superfamily of the low molecular weight binding proteins sharing a basic framework for the binding and transport of small hydrophobic molecules. Determination of the amino terminal sequence of the chicken retinol binding protein excluded identity between this protein and the Ch21.

Amino Acid Sequence↗
PubMed1990

Stabilization of the mRNA follows transcriptional activation of type II collagen gene in differentiating chicken chondrocyte.

The differentiation of chicken endochondral chondrocyte can be induced in vitro by transferring to suspension culture dedifferentiated chondrocytes grown as adherent cells. During the differentiation process, the synthesis of type I collagen is repressed while the genes encoding type II, IX, and X collagens are gradually activated. The changes in the steady-state levels of the mRNAs for type I, IX, and X collagens were shown to be fully accounted for by proportional modifications in the rates of specific gene transcription (Castagnola, P., Dozin, B., Moro, G., and Cancedda, R. (1988) J. Cell Biol. 106, 461-467). In the present report, we demonstrate that the cellular accumulation of type II collagen mRNA in differentiating chondrocyte is regulated not only at the transcription level but also through a stabilization of the RNA transcript as shown by the enhanced half-life of the mRNA in differentiated cells treated with actinomycin D. We also give evidence that this additional mechanism: 1) is specific for type II collagen, 2) does not occur at an early stage of differentiation when type II collagen mRNA starts accumulating, 3) affects only the cytoplasmic form of the RNA transcript.

Animals↗
PubMed1990

Polarized integrin mediates human keratinocyte adhesion to basal lamina.

Epithelial cell interactions with matrices are critical to tissue organization. Indirect immunofluorescence and immunoprecipitations of cell lysates prepared from stratified cultures of human epidermal cells showed that the major integrins expressed by keratinocytes are alpha E beta 4 (also called alpha 6 beta 4) and alpha 2 beta 1. The alpha E beta 4 integrin is localized at the surface of basal cells in contact with the basement membrane, whereas alpha 2 beta 1/alpha 3 beta 1 integrins are absent from the basal surface and are localized only on the lateral surface of basal and spinous keratinocytes. Anti-beta 4 antibodies potently inhibited keratinocyte adhesion to matrigel or purified laminin, whereas anti-beta 1 antibodies were ineffective. Only anti-beta 4 antibodies were able to detach established keratinocyte colonies. These data suggest that alpha E beta 4 mediates keratinocyte adhesion to basal lamina, whereas the beta 1 subfamily is involved in cell-cell adhesion of keratinocytes.

Animals↗
PubMed1990

In vitro development of hypertrophic chondrocytes starting from selected clones of dedifferentiated cells.

Single cells from enzymatically dissociated chick embryo tibiae have been cloned and expanded in fresh or conditioned culture media. A cloning efficiency of approximately 13% was obtained using medium conditioned by dedifferentiated chondrocytes. A cloning efficiency of only 1.4% was obtained when conditioned medium from hypertrophic chondrocytes was used, and efficiencies of essentially 0 were found with fresh medium or medium conditioned by J2-3T3 mouse fibroblasts. Cell clones were selected by morphological criteria and clones showing a dedifferentiated phenotype (fibroblast-like) were further characterized. Out of 38 clones analyzed, 17 were able to differentiate to the hypertrophic chondrocyte stage and reconstitute hypertrophic cartilage when placed in the appropriate culture conditions. Cells from these clones expressed the typical markers of chondrocyte differentiation, i.e., type II and type X collagens. Clones not undergoing differentiation continued to express only type I collagen. Hypertrophic chondrocytes from differentiating clones were analyzed at the single cell level by immunofluorescence; all the cells were positive for type X collagen, while approximately 50% of them showed positivity for type II collagen.

Animals↗
PubMed1990

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. We also demonstrate that this epithelium can be successfully transplanted onto patients presenting lack of adherent keratinizing gingival mucosa and is able, in a relatively short time, to fully express the differentiation program typical of the original donor site. The possibility of obtaining large quantities of cultured epithelium, able to retain properties of the original donor site, starting from 1-3-mm2 biopsies, could prove extremely useful in the reconstructive surgery of the mouth and of other mucosal body areas.

Epithelium↗
PubMed1989

Calcification of in vitro developed hypertrophic cartilage.

We have recently reported that dedifferentiated cells derived from stage 28-30 chick embryo tibiae, when transferred in suspension culture in the presence of ascorbic acid, develop in a tissue closely resembling hypertrophic cartilage. Ultrastructural examination of this in vitro formed cartilage showed numerous matrix vesicles associated with the extracellular matrix (C. Tacchetti, R. Quarto, L. Nitsch, D. J. Hartmann, and R. Cancedda, 1987, J. Cell Biol. 105, 999-1006). In the present article we report that the in vitro developed hypertrophic cartilage undergoes calcification. We indicate a correlation between the levels of alkaline phosphatase activity and calcium deposition at different times of development. Following the transfer of cells into suspension culture and an initial lag phase, the level of alkaline phosphatase activity rapidly increased. In most experiments the maximum of activity was reached after 5 days of culture. When alkaline phosphatase activity and 45Ca deposition were measured in the same experiment, we observed that the increase in alkaline phosphatase preceded the deposition of nonwashable calcium deposits in the cartilage.

Alkaline Phosphatase↗
PubMed1989

Multicentre experience in the treatment of burns with autologous and allogenic cultured epithelium, fresh or preserved in a frozen state.

This report describes the clinical results obtained from a multicentre experience of the use of autologous and allogenic cultured human epidermal cells in the treatment of partial and full skin thickness burns. A laboratory has been organized to supply cultured epithelium to Burns Units in different cities. From May 1986 to December 1988, 58 patients with an age range of 1 to 59 years, and with burns covering between 7 and 95 per cent of the body surface area, have been treated. Graftable cultured epithelium can be frozen and remain viable if stored in a skin bank. Such grafts were used successfully to treat patients with partial and full skin thickness wounds.

Adolescent↗
PubMed1989

The podosomes of Rous sarcoma virus transformed chondrocytes show a peculiar ultrastructural organization.

The ultrastructure of F-actin-containing punctate adhesion structures (podosomes) and of their rosette-like clusters has been studied by transmission electron microscopy in Rous sarcoma virus transformed chick embryo chondrocytes. Peculiar "glove finger" invaginations were found to take origin from the ventral membrane at sites of close contact; they were directed toward the center of the cell perpendicularly from the substratum. These new structures may be the sites where the local release of proteases takes place at the side of cell-to-substratum adhesion in podosome-bearing cells. The cytoplasmic face of glove finger invaginations and of the plasma membrane at cell-to-cell contact is lined by thick accumulations of microfilamentous material.

Animals↗
PubMed1989

Synthesis and secretion of Ch 21 protein in embryonic chick skeletal tissues.

We reported the identification, purification and characterization of a low molecular weight protein (Ch 21) expressed in vitro by differentiating chondrocytes at a late stage of development and observed in vivo in the growth plate region of the long bones at the border between hypertrophic cartilage and newly formed bone (Descalzi Cancedda, F., P. Manduca, C. Tacchetti, P. Fossa, R. Quarto, R. Cancedda, J. Cell Biol. 107, 2455-2463 (1988]. In this article, the synthesis and location of Ch 21 protein in the chick embryo tibia at late stage of development were further investigated. Ch 21 was observed in the cartilage matrix surrounding marrow cavities and in the prearticular outer layer by immunolocalization. In addition, the timing of Ch 21 appearance during the tibia development and its distribution in the growth plate region was better defined. We first observed presence of Ch 21 in the perichondral mid-diaphyseal sleeve of 7-day-old tibia. Ch 21 antibodies stained also the newly formed bone. Synthesis and secretion in the culture medium of Ch 21 protein was observed when bone fragments or cultured osteoblasts isolated from 19-day-old embryo tibiae were labeled in vitro. A search for the presence of Ch 21 in the chick embryo sternum was performed. The synthesis of Ch 21, both in the presumptive calcification cranial portion and in the permanent cartilaginous caudal portion of the sternum, was shown by metabolic labeling of tissue slices.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗