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Nasal reconstruction remains a challenge for any surgeon. The surgical indications for nasal reconstruction after oncologic resection, trauma or as part of cosmetic rhinoplasty, are steadily increasing. The current attitude for reconstruction is the use of autologous cartilage grafts of various origins (septal, ear or rib) trying to restore a physiological anatomy but their quantity is limited. Thus, in order to produce an implantable cartilaginous model, we developed a study protocol involving human nasal chondrocytes, growth factors and a composite biomaterial and studied at the molecular, cellular and tissue level the phenotype of the chondrocytes cultured in this model.After extraction of chondrocytes and their amplification on plastic, the cells were cultured for 15 days either in monolayer or within an agarose hydrogel or a composite biomaterial (agarose/high density polyethylene: Medpor(®)) in the presence or not of a cocktail of soluble factors (BIT): bone morphogenetic protein-2 (BMP-2), insulin and triiodothyronine (T3). The quality of the chondrocyte phenotype was analyzed by PCR, western blotting and immunohistochemistry.During their amplification in monolayer, chondrocytes dedifferentiate. However, our results show that the BIT cocktail induces redifferentiation of chondrocytes cultured in agarose/Medpor with synthesis of mature chondrogenic markers. Thereby, chondrocytes associated with the agarose hydrogel will colonize Medpor and synthesize an extracellular matrix characteristic of nasal cartilage.This nasal cartilage tissue engineering protocol provides the first interesting results for nasal reconstruction.

Despite significant progress in the treatment of glioblastoma, the prognosis of these radioresistant, invasive and hypoxic tumours remain dark. The constant relapse after treatment of this tumour is in part due to its intra-cellular but also micro-environmental radioresistance, largely controlled by growth factors and their receptors. The complexity of the biology of these tumours and the presence of numerous cross-talks between the pathways of these different growth factors can be in part responsible for the negative results obtained in clinical trials associating radiotherapy and targeted drugs designed without previous in vitro and in vivo studies validating the proof of concept of a specific target as key factor of radioresistance. In the aim to optimize the treatment of glioblastoma and to reduce the risks of failure of new trials, several laboratories and clinical departments are developing translational research in radiotherapy and radiobiology, validating in vitro and then in orthotopic xenografts interesting targets, then studying the radiosensitizing effect of targeted drugs directed against these proteins, studying the mechanisms of action and resistance of these drugs, validating these proteins as predictive factors of response to radiotherapy in the patients, and then designing clinical trials, integrating metabolic or functional imaging and surrogate markers to better understand the mechanism of action of these associations. We describe in this article the main translational research axis developed for radiosensitizing glioblastoma, which our lab and department have pursued for several years.

Angiogenesis is central to cancer research. Recent progresses in understanding its mechanisms have enabled the development of therapies that inhibit this process. Many molecules have shown a synergistic effect in combination with irradiation in preclinical studies, and several are being tested in phase I or II. This effect could be explained by a transient normalization of tumor vasculature, leading to improve tumor oxygenation and thus greater radiosensitivity. Although promising, many questions remain about the dose, optimal sequences of the association.

Distraction osteogenesis has become a mainstay in bone engineering and the recent application of this technique to the membranous craniofacial skeleton has significantly improved our armamentarium for reconstructive craniomaxillofacial procedures. However, if the biomechanical, histological and ultrastructural changes associated with distraction osteogenesis have been widely described, the molecular mechanisms governing the formation of new bone in the interfragmental gap of gradually distracted bone segments remain largely unclear. Recently, our laboratory has described a rat mandibular distraction model that provides an excellent environment for deciphering the molecular mechanisms that mediate distraction osteogenesis. In this Article, we present the hypotheses and current research that have furthered our knowledge of the molecular mechanisms that govern distraction osteogenesis. Recent studies have implicated a growing number of cytokines that are intimately involved in the regulation of bone synthesis and turnover. The gene regulation of numerous cytokines (Transforming Growth Factor-B, Bone Morphogenetic Proteins, Insulin-like Growth Factor-1, Fibroblast Growth Factor-2) during distraction osteogenesis have been best characterized and will be discussed in this text. We believe that novel systems like the rat model will facilitate our understanding of the biomolecular mechanisms that mediate membranous distraction osteogenesis and will ultimately guide the development of targeted-strategies designed to accelerate bone healing.

Tumor-associated angiogenesis is emerging as an important prognostic factor and represents a hopeful potential therapeutic target for the cancer treatment. Bladder tumors, as all solid tumors, require an active angiogenesis to support their growth and progression. The angiogenic phenotype observed within a tumor is determined in large part by the balance between stimulatory and inhibitory inputs to the endothelial cells. Clinically, the importance of the angiogenic response observed within a tumor should be considered as an independent prognostic factor for superficial as well as invasive bladder tumors. The analysis of the angiogenic response could in the future influence the therapeutic strategy. Angiogenesis also represents a promising new therapeutic target, and is at the moment intensively tested in experimental and clinical trials. In this article, we will first review the mechanism of angiogenesis, and then its implication in bladder cancer, and as a potential therapeutic target.

Angiogenesis reflects a balance between stimulating and inhibitory factors, among which fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF) play a central role. FGF-2 stimulates endothelial cell growth and migration and enhances angiogenesis, both in vitro and in vivo. FGF-2 binds to a specific receptor (FGF R1) at the surface of endothelial cells. In addition to its extracellular effects, FGF-2 exerts intracellular effects that seem to be independent from tyrosine kinase-type receptors. FGF-2 is produced by several tumor types, including brain tumors, skin tumors, and fibrosarcomas. FGF-2 may play a pivotal role in angiogenesis in these tumors.

The role of FGF-2 in tumor progression and tumor cell invasiveness was investigated using the rat bladder carcinoma cells NBT-II, which do not constitutively express FGF-2 or its membrane-spanning receptor. The NBT-II cells were transfected using expression vectors encoding either the 18 kD or the 24 kD isoform of FGF-2. The 24 kD isoform contains a nuclear localization signal. The transfected NBT-II cells that expressed 18 kD FGF-2 produced and secreted this factor as the biologically active form and retained an epithelial morphology. When injected to nude mice, the tumorigenic potential of these cells was not increased over that of non-transfected NBT-II cells; however, although the time to tumor development was long, the tumors were highly vascularized, indicating secretion of the angiogenic factor FGF-2. The transfected NBT-II cells that expressed 24 kD FGF-2 varied in their morphological appearance and did not secrete FGF-2; immunofluorescence and Western-blot studies showed that the FGF-2 was mainly intranuclear. When injected to nude mice, these cells produced tumors and migrated not only to the lymph nodes but also to the lungs where they produced metastases. In aggregate, these data indicate that stimulation of angiogenesis is not sufficient to increase tumor growth and that nuclear FGF-2 acts as a tumorigenic and metastasis-promoting factor in the NBT-II carcinoma model.

To investigate the influence of fetal calf serum (FCS) and fibroblast growth factor (FGF) on human keratocyte growth in vitro and cell differentiation, and to describe cultured human keratocyte ultra-structure.Human keratocytes were cultured in TC 199/Ham F12 media, supplemented or not with 10% FCS, aFGF, and bFGF. Keratocyte growth was studied. Cultured keratocytes were analyzed by means of immunochemistry and transmission electron microscopy.Without fetal calf serum, cell population doubling occurred after 7 days of culture and no alpha smooth muscle-actin cell expression was observed. With serum, cell population increased by 1 log after 7 days of culture and all of the cells were alpha SM-actin + bFGF or aFGF-addition to the serum-containing medium resulted in a dramatic decrease in this alpha SM-actin expression. Nuclei were found to be oval and regular in cross-sections, and round and indented in frontal sections. Numerous cytoplasmic organelles were observed, as were cell expansions, gap junctions, omega-shaped structures, and fenestrations. Cultured keratocytes synthesized collagen fibers and filaments.Fetal calf serum allows human keratocytes to grow with a myofibroblast cell phenotype, whereas addition of FGF results in a fibroblast cell phenotype. Ultrastructure of cultured keratocyte is similar to that observed in situ.

Hemangiomas of infancy follow a characteristic three-phases course: proliferation, involution, regressed Proliferative endothelial cells predominate during the proliferative phase. Moreover it has been shown that patients with active angiogenesis have elevated levels of urinary bFGF (basic Fibroblast Growth Factor).Here we report our preliminary results of urinary bFGF assay (ELISA) for the diagnosis and follow up of severe hemangioma. We also assayed bFGF in normal infants, in patients with large vascular malformations and in infants with Kasabach-Merritt syndrome.In the control group, urinary bFGF was elevated in new borns but nul or very low in infants. Urinary bFGF levels were normal, i.e. very low in 4 patients with a vascular malformation. In infants with a clinically proliferative hemangioma, urinary bFGF was elevated in 8 among the 10 studied. bFGF levels guided treatment in 9 patients. Urinary bFGF was elevated in 4 patients with Kasabach-Merritt syndrome.Angiogenesis is regulated by angiogenic and inhibitory factors. The angiogenic factor bFGF is an autocrine growth factor for endothelial cells and hemangioma endothelial cells expressing bFGF in their cytosol during the proliferative phase. As suggested by J. Folkman and his group, assay of urinary bFGF appears useful in differentiating between hemangioma and vascular malformation and for follow up of treated patients.

Two-dimensional electrophoresis analysis of proteins from breast cancer cells MCF-7. Modifications of synthesis induced by FGF-2. Using high resolution two-dimensional electrophoresis, we have separated more than 1,000 proteins from the breast cancer cell line MCF-7. Computer assisted analysis of gels allowed us to classify these proteins in function of their isoelectric point, molecular weight and relative quantity. This data-base will now be used as a powerful tool to identified proteins which synthesis is regulated in various experimental or pathological situations. Thus we studied modifications induced by FGF-2. This growth factor induces the synthesis of four polypeptides which are not detected in cells not stimulated by this factor. In addition, intensity of nine other polypeptides was found increased in presence of FGF-2.

Regulation of proliferation and migration are well known roles of fibroblast growth factor 2 (FGF-2) for arterial smooth muscle cells (SMC). We show here, by sense cDNA transfection that endogenous FGF-2 expression controls alpha-actin level in SMC clones. All the high alpha-actin expressing clones were FGF-2 transfected. Control clones carrying a deleted vector showed a weak expression and an altered actin polymerisation compared to the parental cultures. Among FGF-2 transfected clones, alpha-actin expression was heterogenous with diversely high levels. These observations were obtained using normal rat SMC or SMC from a transformed cell line. They indicate a role for endogenous FGF-2 in arterial SMC differentiation. Our results suggest that FGF-2 might act either by permissing clonal growth of already differentiated cells or by regulating expression or stability of alpha-actin. They open new perspective for gene therapy of the arterial wall.

The MCF-7 breast cancer cells exhibit remarkable growth enhancement in response to basic fibroblast growth factor (FGF-2) stimulation in a dose dependent manner. To investigate the involvement of proteoglycans on control of FGF-2 induced proliferation, polysaccharide chains were degraded by specific enzymes. Our results showed that MCF-7 cells were unsensitive to FGF-2 after enzymatic degradation of heparin sulfate proteoglycans (HSPG) by heparinase. After metabolic inhibition of sulphation by sodium chloride, radiolabelled proteoglycans were purified and quantified by ion exchange chromatography. Sodium chlorate treatment reduced by 70% sulfation of proteoglycans. This decrease of sulphation totally inhibited FGF-2-mediated proliferation. The sulphated glycosaminoglycans which were critical in FGF-2-induced proliferation were strictly HSPG, as an addition of heparin in cell culture medium can restore FGF-2 mitogenic activity. In contrast, other glycosaminoglycans (chondroitin sulfate/hyaluronic acid) did not show any effect. These results provide clear evidence for the critical role of HSPG in FGF-2-induced proliferation on MCF-7 breast cancer cells.

Administration at doses of 1,800 ng to 6,200 ng of basic fibroblast growth factor (bFGF), around the posterior limb buds of young slow-worm (Anguis fragilis, L.) embryos developing in cultured eggs has, after 4 to 6 days, in several embryos (10 among 15, treated at stage of allantoic bud, from 3 to 9 mm long), delayed the involution of these posterior limbs: their anlagen are larger than the control ones and histological examination shows an increase, between 20% and 30%, of cell density in the mesodermal component of the limb bud. These results bring into light the possibility to control, to some degree, the regressive evolution of the limbs.

Administration at doses of 1,800 ng to 6,200 ng of basic fibroblast growth factor (bFGF), around the posterior limb buds of young slow-worm (Anguis fragilis, L.) embryos developing in cultured eggs has, after 4 to 6 days, in several embryos (10 among 15, treated at stage of allantoic bud, from 3 to 9 mm long), delayed the involution of these posterior limbs: their anlagen are larger than the control ones and histological examination shows an increase, between 20% and 30%, of cell density in the mesodermal component of the limb bud. These results bring into light the possibility to control, to some degree, the regressive evolution of the limbs.

Tenascin (TN) is an extracellular matrix (ECM) glycoprotein which possesses antiadhesive properties and thus is able to modulate cell interactions with molecules of the ECM. In Xenopus embryos, TN is expressed dorsally in a very restricted pattern. We have studied the distribution of TN mRNA in tailbud-stage embryos by in situ hybridization. We show that TN transcripts are principally expressed in myotome cells. No TN mRNA could be detected in lateral plate mesoderm. This had led us to study TN expression in response to mesodermal inducers. Analysis of the distribution of TN after mesodermal induction of blastula animal caps with activin A and bFGF or after ectopic expression of XBra shows that TN expression is elicited when dorsal or posterior structures are formed. Our results show that TN regionalization in Xenopus embryos depends on mesoderm patterning.

Culture techniques for normal human melanocytes have been developed over the last ten years. This in vitro model for studying pigment-producing cells can be expected to provide major advances in the knowledge of cell-to-cell anc cell-to-matrix interactions, melanocyte and melanin biology, pathophysiology of pigment disorders, and malignant melanomas. Melanocyte cultures have already shed new light on keratinocyte-melanocyte interactions within the epidermal melanin unit by showing that keratinocytes produce "melanotrophic factors" which modulate growth, melanin production, and dendricity of melanocytes. Melanocyte cultures also enable in vitro studies of melanocyte responses to ultraviolet radiations and of the biologic messengers involved in these responses. Lastly, they provide a means for investigating endocrine and paracrine mechanisms involved in the regulation of melanogenesis, including the role of melanotropins, estrogens and vitamin D. Improved knowledge of the molecular biology of melanocytes provides hope for rapid advances in the understanding of tyrosinase and posttyrosinase regulation of melanogenesis. Lastly, melanocyte cultures can be expected to find useful applications in the pathophysiologic study of pigment disorders and of pharmacologic modulation of skin pigmentation.

We investigated binding characteristics of bFGF in membranes prepared from 4 human breast cancer cell lines (MCF-7, T-47D, BT-20 and MDA-MB-231) and 38 primary breast cancer biopsies. Results of competitive binding experiments were analysed using the "Ligand" program to determine binding site concentrations and affinities. bFGF mitogenic activity was also measured by [3H]-thymidine incorporation into DNA of breast cancer cell lines. The presence of high-affinity binding sites was demonstrated in each cell type (Kd: 0.5 nM). The presence of these high-affinity binding sites was confirmed by saturation experiments. A second class of low-affinity binding sites was detected in the 2 hormono-independent cells (BT-20: Kd = 2.9 nM; MDA-MB-231: Kd = 2.7 nM). bFGF stimulated the proliferation of MCF-7, 7-47D, BT-20 and not of MDA-MB-231 cell lines. In breast cancer biopsies, binding sites were detectable in 36/38 cases; high-affinity binding sites (Kd1 nM) were present in 19/39 cases and low-affinity binding sites (Kd2 nM) were present in 29/36 cases (the 2 classes of binding sites were present in 12 biopsies). No relation between FGF binding sites and node involvement nature or grade of tumor was evidenced. Negative correlations (Spearman test) were found between total bFGF binding site concentrations and estradiol receptor concentrations (P = 0.05) or progesterone receptor concentrations (P = 0.009). The demonstrations of 1), bFGF specific binding sites in breast cancer membranes; and 2) bFGF growth stimulation of some breast cancer cell lines, indicate that this factor could be involved in the growth of most breast cancers, and could act (among other factors) directly on the growth of cancer cells.

Basic fibroblast growth factor (bFGF or FGF-2) is present in the basal membrane of pancreatic cells during the pancreatic embryonic development. The expression of bFGF receptors has been described in normal pancreatic cells. By contrast, pancreatic cancer cells express not only the bFGF receptors but also the bFGF itself. With the aim of understanding the effects induced by the production of bFGF by pancreatic cancer cells, the pancreatic acinar cell line (AR4-2J) was used. AR4-2J cells do not produce bFGF but express bFGF receptors. These cells were transfected with a vector containing the bFGF cDNA encoding the three different forms of bFGF characterized in tumor cells. Results showed that the bFGF expression induced important phenotypic and enzymatic modifications. The transfected cells lost some morphological features of the acinar cells and expressed amylase and lipase at low levels (a 90% decrease for amylase activity, whereas lipase activity was barely detectable). These results suggest that bFGF could be involved in maintaining pancreatic cells in a slightly differentiated state.

Vasculotropin is a growth factor with a unique specificity for vascular derived endothelial cells. We report that normal human peripheral lymphocytes represent another target for vasculotropin. The mitogenic activity of the medium conditioned by these cells cultured in the presence of Concanavalin A is potentiated by vasculotropin. This effect is exerted more likely through interactions with the soluble mediators rather than through the VAS receptors since VAS binds equally to Concanavalin A stimulated and to unstimulated lymphocytes.