Ranieri Cancedda - Academia.edu (original) (raw)

Papers by Ranieri Cancedda

Frontiers in Bioengineering and Biotechnology

Maintenance of tissue homeostasis and tissue regeneration after an insult are essential functions... more Maintenance of tissue homeostasis and tissue regeneration after an insult are essential functions of adult stem cells (SCs). In adult tissues, SCs proliferate at a very slow rate within “stem cell niches”, but, during tissue development and regeneration, before giving rise to differentiated cells, they give rise to multipotent and highly proliferative cells, known as transit-amplifying cells (TACs). Although differences exist in diverse tissues, TACs are not only a transitory phase from SCs to post-mitotic cells, but they also actively control proliferation and number of their ancestor SCs and proliferation and differentiation of their progeny toward tissue specific functional cells. Autocrine signals and negative and positive feedback and feedforward paracrine signals play a major role in these controls. In the present review we will consider the generation and the role played by TACs during development and regeneration of lining epithelia characterized by a high turnover including...

Journal of Biological Chemistry, 1996

The Journal of Cell Biology, 1987

Dedifferentiated chick embryo chondrocytes (Castagnola, P., G. Moro, F. Descalzi-Cancedda, and R.... more Dedifferentiated chick embryo chondrocytes (Castagnola, P., G. Moro, F. Descalzi-Cancedda, and R. Cancedda, 1986, J. Cell Biol., 102:2310-2317), when transferred to suspension culture on agarose-coated dishes in the presence of ascorbic acid, aggregate and remain clustered. With time in culture, clusters grow in size and adhere to each other, forming structures that may be several millimeters in dimension. These structures after 7 d of culture have the histologic appearance of mature hypertrophic cartilage partially surrounded by a layer of elongated cells resembling the perichondrium. Cells inside the aggregates have ultrastructural features of stage I (proliferating) or stage II (hypertrophic) chondrocytes depending on their location. Occurrence and distribution of type I, II, and X collagens in the in vitro-formed cartilage at different times of culture, show a temporal and spatial distribution of these antigens reminiscent of the maturation events occurring in the cartilage in v...

Endocrinology, 1997

The factors regulating the growth and development of mesenchymal precursor cells toward chondroge... more The factors regulating the growth and development of mesenchymal precursor cells toward chondrogenesis are not well identified. We have developed a defined serum-free culture system that allows the proliferation of chick embryo chondrogenic cells and their maturation toward hypertrophic chondrocytes. Proliferation is obtained in adhesion in medium supplemented with insulin (Ins), Dexamethasone (Dex), and either basic fibroblast growth factor (FGF-2), plateletderived growth factor bb, epithelial growth factor, or GH; the highest mitogenic response is induced by FGF-2 in synergy with Ins. Ins can be substituted by Ins-like growth factor I. When these cells are transferred into suspension culture in Ins/Dex and T 3 without growth factor supplement, they undergo the complete chondrogenic development characterized by type X collagen synthesis and cellular hypertrophy. During differentiation, Ins cannot be substituted by Inslike growth factor I. Chondrogenesis is also evidenced by the formation of hypertrophic cartilage when the medium is supplemented with ascorbic acid. If T 3 is introduced in the proliferation phase, the cells fail to differentiate to hypertrophy in suspension unless bone morphogenetic protein-2 is added. Assays of ectopic tissue formation in nude mice, with cells implanted sc after adsorption on collagen sponge or porous hydroxyapatite ceramics, indicate that cells grown in Ins/FGF-2 reform mainly cartilage in vivo, whereas expansion in Ins/T 3 /Dex/FGF-2 leads to the formation of cartilage, bone, and adipose tissue.

During vertebrate embryogenesis, bones of the vertebral column, pelvis, and upper and lower limbs... more During vertebrate embryogenesis, bones of the vertebral column, pelvis, and upper and lower limbs, are formed on an initial cartilaginous model. This process, called endochondral ossification, is characterized by a precise series of events such as aggregation and differentiation of mesenchymal cells, and proliferation, hypertrophy and death of chondrocytes. Bone formation initiates in the collar surrounding the hypertrophic cartilage core that is eventually invaded by blood vessels and replaced by bone tissue and bone marrow. Over the last years we have extensively investigated cellular and molecular events leading to cartilage and bone formation. This has been partially accomplished by using a cell culture model developed in our laboratory. In several cases observations have been confirmed or directly made in the developing embryonic bone of normal and genetically modified chick and mouse embryos. In this article we will review our work in this field.

Journal of Cell Science, 1997

A subtracted cDNA library was generated to identify cDNAs specific for chondrocyte mRNAs preferen... more A subtracted cDNA library was generated to identify cDNAs specific for chondrocyte mRNAs preferentially expressed at the hypertrophic stage with respect to early differentiation stages. The characterization of a cDNA isolated from this library that hybridizes with a 1.8 kb mRNA is described here. This mRNA is expressed at extremely low levels in dedifferentiated chondrocytes cultured in adherent conditions, at very low levels in differentiating chondrocytes and at very high levels in hypertrophic chondrocytes cultured in suspension conditions. In the developing chick embryo this mRNA is detectable in RNAs extracted from several other tissues besides cartilage. The described cDNA contains a complete open reading frame coding for a polypeptide of about 33 kDa. Homology searches with known cDNA and protein sequences have revealed that the chicken protein is related to the amino-terminal half of two mammalian nuclear antigens. By immunohistochemistry with specific rabbit antisera a stro...

Journal of Cell Science, 2001

Avidin is a major [(35)S]methionine-labeled protein induced by bacterial lipopolysaccharide (LPS)... more Avidin is a major [(35)S]methionine-labeled protein induced by bacterial lipopolysaccharide (LPS) and interleukin 6 (IL-6) in cultured chick embryo myoblasts and chondrocytes. It was identified by N-terminal sequencing of the protein purified from conditioned culture medium of LPS-stimulated myoblasts. In addition, avidin was secreted by unstimulated myoblasts and chondrocytes during in vitro differentiation; maximal expression being observed in differentiated myofibers and hypertrophic chondrocytes. In developing chick embryos, immunohistochemistry revealed avidin in skeletal muscles and growth plate hypertrophic cartilage. Avidin was secreted into culture as a biologically active tetramer. Exogenous avidin added to the medium of proliferating chondrocytes progressively inhibited cell proliferation, whereas addition of avidin to differentiating chondrocytes in suspension allowed full cell differentiation. No toxic effects for the cells were observed in both culture conditions. West...

Journal of Cell Science, 2000

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) induces endothelial ce... more Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) induces endothelial cell migration and proliferation in culture and is strongly angiogenic in vivo. VEGF synthesis has been shown to occur in both normal and transformed cells. The receptors for the factor have been shown to be localized mainly in endothelial cells, however, the presence of VEGF synthesis and the VEGF receptor in cells other than endothelial cells has been demonstrated. Neoangiogenesis in cartilage growth plate plays a fundamental role in endochondral ossification. We have shown that, in an avian in vitro system for chondrocyte differentiation, VEGF was produced and localized in cell clusters totally resembling in vivo cartilage. The factor was synthesized by hypertrophic chondrocytes and was released into their conditioned medium, which is highly chemotactic for endothelial cells. Antibodies against VEGF inhibited endothelial cell migration induced by chondrocyte conditioned media. Similarly...

Development, 1993

Dedifferentiated chondrocytes cultured adherent to the substratum proliferate and synthesize larg... more Dedifferentiated chondrocytes cultured adherent to the substratum proliferate and synthesize large amounts of type I collagen but when transferred to suspension culture they decrease proliferation, resume the chondrogenic phenotype and the synthesis of type II collagen, and continue their maturation to hypertrophic chondrocyte (Castagnola et al., 1986, J. Cell Biol. 102, 2310–2317). In this report, we describe the developmentally regulated expression of type VI collagen in vitro in differentiating avian chondrocytes. Type VI collagen mRNA is barely detectable in dedifferentiated chondrocytes as long as the attachment to the substratum is maintained, but increases very rapidly upon passage of the cells into suspension culture reaching a peak after 48 hours and declining after 5–6 days of suspension culture. The first evidence of a rise in the mRNA steady-state levels is obtained already at 6 hours for the alpha 3(VI) chain. Immunoprecipitation of metabolically labeled cells with type...

Frontiers in bioengineering and biotechnology, 2017

Present cell culture medium supplements, in most cases based on animal sera, are not fully satisf... more Present cell culture medium supplements, in most cases based on animal sera, are not fully satisfactory especially for the in vitro expansion of cells intended for human cell therapy. This paper refers to (i) an heparin-free human platelet lysate (PL) devoid of serum or plasma components (v-PL) and (ii) an heparin-free human serum derived from plasma devoid of PL components (Pl-s) and to their use as single components or in combination in primary or cell line cultures. Human mesenchymal stem cells (MSC) primary cultures were obtained from adipose tissue, bone marrow, and umbilical cord. Human chondrocytes were obtained from articular cartilage biopsies. In general, MSC expanded in the presence of Pl-s alone showed a low or no proliferation in comparison to cells grown with the combination of Pl-s and v-PL. Confluent, growth-arrested cells, either human MSC or human articular chondrocytes, treated with v-PL resumed proliferation, whereas control cultures, not supplemented with v-PL, ...

Frontiers in Bioengineering and Biotechnology, 2017

For repair of chronic or difficult-to-heal tissue lesions and defects, major constraints exist to... more For repair of chronic or difficult-to-heal tissue lesions and defects, major constraints exist to a broad application of cell therapy and tissue engineering approaches, i.e., transplantation of "ex vivo" expanded autologous stem/progenitor cells, alone or associated with carrier biomaterials. To enable a large number of patients to benefit, new strategies should be considered. One of the main goals of contemporary regenerative medicine is to develop new regenerative therapies, inspired from Mother Nature. In all injured tissues, when platelets are activated by tissue contact, their released factors promote innate immune cell migration to the wound site. Platelet-derived factors and factors secreted by migrating immune cells create an inflammatory microenvironment, in turn, causing the activation of angiogenesis and vasculogenesis processes. Eventually, repair or regeneration of the injured tissue occurs via paracrine signals activating, mobilizing or recruiting to the wound site cells with healing potential, such as stem cells, progenitors, or undifferentiated cells derived from the reprogramming of tissue differentiated cells. This review, largely based on our studies, discusses the identification of new tools, inspired by cellular and molecular mechanisms overseeing physiological tissue healing, that could reactivate dormant endogenous regeneration mechanisms lost during evolution and ontogenesis.

Frontiers in Immunology, 2016

The Journal of Cell Biology, 1986

In the developing chick embryo tibia type X collagen is synthesized by chondrocytes from regions ... more In the developing chick embryo tibia type X collagen is synthesized by chondrocytes from regions of hypertrophy and not by chondrocytes from other regions (Capasso, O., G. Tajana, and R. Cancedda, 1984, Mol. Cell. Biol. 4:1163-1168; Schmid, T. M., and T. F. Linsenmayer, 1985, Dev. Biol. 107:375-381). To investigate further the relationship between differentiation of endochondral chondrocytes and type X collagen synthesis we have developed a novel culture system for chondrocytes from 29-31-stage chick embryo tibiae. At the beginning of the culture these chondrocytes are small and synthesize type II and not type X collagen, but when grown on agarose-coated dishes they further differentiate into hypertrophic chondrocytes that synthesize type X collagen. The synthesis of type X collagen has been monitored in cultured cells by analysis of labeled collagens and in vitro translation of mRNAs. When the freshly dissociated chondrocytes are plated in anchorage-permissive dishes, most of the c...

The Journal of Cell Biology, 1990

Single cells from enzymatically dissociated chick embryo tibiae have been cloned and expanded in ... more 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...

The Journal of Cell Biology, 1988

When transferred to suspension culture on agarose-coated dishes, dedifferentiated chick embryo ch... more When transferred to suspension culture on agarose-coated dishes, dedifferentiated chick embryo chondrocytes resume the chondrocyte phenotype and continue their maturation to hypertrophic chondrocytes (Castagnola, P., G. Moro, F. Descalzi Cancedda, and R. Cancedda. 1986. J. Cell Biol. 102:2310-2317). In this paper we report the identification, purification, and characterization of a low molecular weight protein, named Ch 21, expressed and secreted by in vitro differentiating chondrocytes at a late stage of development. This protein is detectable in the cells after a short pulse labeling and is directly secreted in the culture medium. The Ch 21 protein has a peculiar resistance to limited pepsin digestion; nevertheless it is not collagenous in nature as revealed by its unaltered mobility when isolated from cells grown in the presence of alpha-alpha' dipyridyl, its resistance to bacterial collagenase, and its amino acid composition. By metabolic labeling of tissue slices and by imm...

Journal of Cell Biology, 1992

Chondrocytes from chicken embryo tibia can be maintained in culture as adherent cells in Coon&#39... more 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. Differentiation is evidenced by the shift from type I to type II and type IX collagen synthesis and the following predominant expression of type X collagen, all markers of specific stages of the differentiation process. To identify the factors required for differentiation, we developed a serum-free culture system where only the addition of triiodothyronine (T3; 10(-11) M), insulin (60 ng/ml), and dexamethasone (10(-9) M) to the F-12 medium was sufficient to obtain hypertrophic chondrocytes. In this hormonal context, chondrocytes display the same changes in the pattern of protein synthesis as described above. For proper ...

Osteoarthritis and Cartilage, 2005

Objective: Proliferation and chondrogenic commitment of cultured articular chondrocytes are impai... more Objective: Proliferation and chondrogenic commitment of cultured articular chondrocytes are impaired when cells derive from aged donors. In those subjects the feasibility of cell-based therapies for articular surface repair is reduced. Moreover, the use of serum as medium supplement elicits non-physiological responses in cultured chondrocytes. This study was therefore undertaken to identify the expansion culture conditions needed to sustain growth and chondrogenic commitment of chondrocytes harvested from aged human subjects. Design: Articular cartilage was obtained from aged (69e75 years) and from young adult subjects (27e35 years). Chondrocytes were isolated and cultured in serum-free (SF) or in serum-supplemented [fetal calf serum (FCS)] conditions. Chondrocytes were expanded in monolayer for five duplications and processed for RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The differentiation potential was assessed by micromass pellet cultures before and after expansion in either culture medium, or after a prolonged exposure to serum followed by a period in SF condition. Results: Only SF-cultured chondrocytes reached five duplications within 25e35 days, maintaining the expression of some chondrogenic markers and without altering the levels of active matrix metalloproteinase 3 (MMP-3). Only the pellets derived from SF-expanded cultures positively stained for cartilage matrix deposition. On the contrary, exposure to serum diminished the proliferation capacities, abolished the differentiation potential in the same cells and elicited transcription of the MMP-3 gene. Shifting culture conditions from FCS to SF resumed growth rates but proper extracellular matrix deposition was only partially restored. Conclusions: The SF conditions have proven valuable to prime cell proliferation and to sustain proper commitment in chondrocytes from aged patients. This culturing approach may represent a therapeutic chance extendable to a range of patients normally excluded from clinical protocols based on autologous chondrocyte implantation (ACI).

Osteoarthritis and Cartilage, 2013

Objective: Studies about cartilage repair in the hip and infant chondrocytes are rare. The aim of... more Objective: Studies about cartilage repair in the hip and infant chondrocytes are rare. The aim of our study was to evaluate the use of infant articular hip chondrocytes for tissue engineering of scaffold-assisted cartilage grafts. Method: Hip cartilage was obtained from five human donors (age 1e10 years). Expanded chondrocytes were cultured in polyglycolic acid (PGA)-fibrin scaffolds. De-and re-differentiation of chondrocytes were assessed by histological staining and gene expression analysis of typical chondrocytic marker genes. In vivo, cartilage matrix formation was assessed by histology after subcutaneous transplantation of chondrocyte-seeded PGA-fibrin scaffolds in immunocompromised mice. Results: The donor tissue was heterogenous showing differentiated articular cartilage and nondifferentiated tissue and considerable expression of type I and II collagens. Gene expression analysis showed repression of typical chondrocyte and/or mesenchymal marker genes during cell expansion, while markers were re-induced when expanded cells were cultured in PGA-fibrin scaffolds. Cartilage formation after subcutaneous transplantation of chondrocyte loaded PGA-fibrin scaffolds in nude mice was variable, with grafts showing resorption and host cell infiltration or formation of hyaline cartilage rich in type II collagen. Addition of human platelet rich plasma (PRP) to cartilage grafts resulted robustly in formation of hyaline-like cartilage that showed type II collagen and regions with type X collagen. Conclusion: These results suggest that culture of expanded and/or de-differentiated infant hip cartilage cells in PGA-fibrin scaffolds initiates chondrocyte re-differentiation. The heterogenous donor tissue containing immature chondrocytes bears the risk of cartilage repair failure in vivo, which may be possibly overcome by the addition of PRP.

Journal of Tissue Engineering and Regenerative Medicine, 2009