[Replacement of the trachea using surgical reconstruction: current state of research]. / Remplacement de trachée par reconstructions chirurgicales: état actuel de la recherche.

OBJECTIVE: To review the main studies and the recent surgical procedures in tracheal reconstruction. MATERIAL AND METHOD: The literature search was conducted using the key words "tracheal reconstruction", "grafts", and "tissue engineering" and by selecting references from the articles reviewed as well as the experience of the authors in this field. RESULTS: Surgical reconstruction for tracheal replacement without using biomaterials involves tissue grafts (auto- or allografts) and tissue engineering. Among the many procedures already described, three new techniques have emerged these past few years employing autologous mesenchymal stem-cell-derived chondrocytes, autologous cultured epithelial cells, and a matrix derived from tracheal graft; costal cartilage, recipient mucosa, and local or free flaps, and an aortic graft. These procedures have been proposed in humans with apparently good results but with a still limited follow-up. CONCLUSIONS: Tracheal reconstruction techniques have recently progressed and replacing a long segment of trachea can be envisaged for the future. Moreover, these reconstructions, in conjunction with biomaterial development, would facilitate the design and the implantation of a laryngeal prosthesis.

[Reconstruction of the anterior mandibule using a porous titanium implant: a case report]. / Reconstruction de la symphyse mandibulaire par implant de titane poreux: à propos d'un cas.

OBJECTIVE: Anterior mandibular arch reconstruction. PATIENT AND METHOD: A 55-year-old immuno-depressed female underwent resection of the lower third of the face subsequent to extensive mucormycosis-related necrosis. Reconstruction of the anterior part of the mandible and adjacent soft tissue was carried out with a mandibular prosthesis and a latissimus dorsi flap. The mandibular prosthesis was made of titanium T40 micro-beads, consolidated by two parallel plates of titanium. The porous structure is intended to enhance cellular and bone integration. RESULTS: The tolerance of the prosthesis was still excellent after 36 months. Labial continence was restored in a second procedure. Unrestricted diet was allowed from the third month. Radiological investigations confirm the good stability of the prosthesis. CONCLUSION: This original prosthesis design offers an alternative to the reconstruction of an anterior mandibular arch by plate or by vascularised free osseous tissue transfer that is sometimes associated with significant morbidity.

Development of tracheal prostheses made of porous titanium: a study on sheep.

Authors report the development of a biomaterial to be used for tracheal and laryngeal reconstruction. This experimentation follows the replacement of trachea in rats with porous titanium implants. The aim of the study is to test this type of prosthesis on sheep, whose trachea is of comparable size to that of humans. Six ewes were implanted with porous titanium implants after resection of 5 cm of trachea. The planned period for the implantation was from 3 to 6 months before the sacrifice of the animals for histological analysis. After a simple immediate postoperative course, the implantations developed complications of tracheal patency, responsible for four deaths (tracheal obstruction by mucous plug n = 2, inferior necrosis of trachea n = 1, pneumopathy n = 1). The two remaining sheep presented no complications. The mechanical performance of the prostheses was good. The histological results showed an inflammatory stenosis of the tracheo-prosthetic junctions, which was not the direct result of death. The protheses were integrated by the surrounding tissue, but endoprosthetic colonisation by pseudostratified ciliated columnar epithelium was low or nil. The absence of endoprosthetic lining was responsible for the complications. The biocompatibility of the biomaterial is not in question, but the surgical procedure will have to be modified by an endoprosthetic mucous graft before implantation so as to accelerate healing process.

Initial adhesion of endothelial cells on polyelectrolyte multilayer films.

Polyelectrolyte multilayer films were recently investigated to favour attachment of Human Vein Umbilical Endothelial Cells (HUVECs) on non-adhesive surfaces. In this study, we evaluated the initial adhesion of HUVECs after 3 h of seeding on two polyelectrolyte multilayer films ending by poly(D-lysine) (PDL) or poly(allylamine hydrochloride) (PAH). In order to obtain information about initial adhesion of HUVECs, cell morphology as well as the expression of beta1 integrins, specific receptors of adhesion, were evaluated after 3 h of seeding on polyelectrolyte multilayer films. The data were also compared to PDL or PAH monolayers (polyelectrolytes terminating the multilayer architecture). The expression of beta1 integrins was not different, whatever are the studied surfaces. However, HUVECs spreading on polyelectrolyte multilayer films, in particular on PAH ending film, was more important as compared to polyelectrolyte monolayers or glass. In conclusion, the best initial adhesion conditions of HUVECs on polyelectrolyte films could not be elucidated, moreover the results suggested also that beta1 integrins could only play a limited role.

Experimental study of a porous rat tracheal prosthesis made of T40: long-term survival analysis.

In order to repair large defects in the laryngotracheal area, we developed a biomaterial based on porous titanium (Ti40) formed of spherical particles that are welded together. These Ti40 beads were arranged in several layers to create the rat tracheal prosthesis. After a partial tracheal resection, the prosthesis was fixed to both extremities to replace the missing part. Tissue surrounding the prosthesis was collected from 33 surviving animals after an implantation period of 3 to 12 months. Histological analyses showed that the periphery of the prosthesis was covered with fibroblasts and a few lymphocytes that penetrated the titanium layers. A ciliary cylindrical epithelium of respiratory type was found on the endoluminal side. The inflammatory reaction observed was minimal. These data indicate that the prosthesis, implanted in a laryngotracheal environment, is well tolerated by animals. Our results represent the first step towards the construction of a total laryngeal prosthesis that should allow restoration of the essential functions of the larynx after a laryngectomy in cancer treatment.

Cell interactions with polyelectrolyte multilayer films.

The short-term interactions of chondrosarcoma cells with polyelectrolyte multilayer films built up by the alternate adsorption of poly(L-lysine) (PLL) and poly(L-glutamic acid) (PGA) was studied in the presence and in the absence of serum. The films and their interaction with serum proteins were first characterized by means of optical waveguide lightmode spectroscopy, quartz crystal microbalance, and zeta potential measurements. In a serum-containing medium, the detachment forces measured by the micropipet technique were about eight times smaller on PGA-ending than on PLL-ending films. For these latter ones, the adhesion force decreased when the film thickness increased. In a serum-free medium, the differences between the negative- and positive-ending films were enhanced: adhesion forces on PLL-ending films were 40-100% higher, whereas no cellular adherence was found on PGA-terminating films. PGA-ending films were found to prevent the adsorption of serum proteins, whereas important protein adsorption was always observed on PLL-ending films. These results show how cell interactions with polyelectrolyte films can be tuned by the type of the outermost layer, the presence of proteins, and the number of layers in the film.

Transcription-dependent nucleocytoplasmic distribution of hnRNP A1 protein in early mouse embryos.

A unique feature of certain members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family of proteins is that they shuttle continuously between nucleus and cytoplasm and their accumulation in the nucleus is transcription-dependent. An extensively characterised protein of this group is hnRNP A1. To date, most studies addressing the transcription-dependent transport of hnRNP A1 have been performed on cultured cell lines treated with transcription inhibitors. Here we have analysed the nucleocytoplasmic distribution of hnRNP A1 in early mouse embryos, where the haploid pronuclei remain transcriptionally inactive for a period of several hours. Consistent with its small molecular size (36 kDa), the hnRNP A1 protein diffuses passively through the nuclear pores and equilibrates between the nucleus and the cytoplasm of transcriptionally inactive embryos. In contrast, following transcriptional activation the A1 protein becomes accumulated in the nucleus. This accumulation of the A1 protein in the nucleus is blocked by the lectin wheat germ agglutinin (WGA), which binds to nuclear pore proteins and prevents translocation of receptor-cargo complexes through the pores. This indicates that a carrier-mediated transport pathway is required for the concentration of A1 in transcriptionally active nuclei. To further analyse how transcription is coupled to nucleocytoplasmic transport, we transplanted transcriptionally inactive pronuclei into the cytoplasm of transcriptionally active embryos. The results show that the presence of newly synthesised RNAs in the cytoplasm is not sufficient to induce the accumulation of hnRNP A1 in the nucleus. Rather, the appearance of nascent transcripts in the nucleus appears to be the crucial event. Since hnRNP A1 is a shuttling protein, an increase in its steady state nuclear concentration could be the result of either faster nuclear import or slower export to the cytoplasm. We propose that binding of A1 to nascent transcripts retards its export to the cytoplasm and therefore contributes to its concentration in the nucleus.

Development-dependent localization of nuclear antigens in growing mouse oocytes.

We have analyzed the distribution of nuclear and nucleolar proteins during the period of oocyte's growth. Oocytes were isolated mechanically or enzymatically from ovaries of juvenile mice of various ages (from 1 to 28 days after birth). Small nuclear ribonucleoproteins (snRNPs), the splicing factor SC-35, and a protein linked to cell proliferation (p-120) were detected by indirect immunofluorescence. snRNP distribution is consistent with the prophase state of oocyte's nuclei, while SC-35 (and p-120) exhibit a "speckled" distribution throughout the entire period of growth. The number of speckles (or foci) appears maximal around 10 days after birth, i.e., in the period of maximal transcriptional activity, and is sensitive to alpha-amanitin treatment. On the other hand, the immunofluorescent distribution of of nucleolin and p-103 (a nucleolar marker of the granular component) is compared to the ultrastructural distribution of the granular component analyzed by electron microscopy on oocytes of the same age.

Redistribution of nuclear antigens linked to cell proliferation and RNA processing in mouse oocytes and early embryos.

We have systematically analyzed by indirect immunofluorescence the subcellular distribution of nuclear antigens in relation to developmental stages of maturing mouse oocytes and developing embryos. Antigens were of two types: (1) a protein whose nuclear localization in interphase somatic cells depends on their proliferative state protein recognized by a monoclonal antibody 43B1N, and (2) snRNP polypeptides recognized by autoimmune sera of anti-Sm and anti-RNP type. The protein recognized by 43B1N was present in the germinal vesicle of oocytes from antral follicles, but absent from the nuclei during the first hours of embryonic life up to the middle to late 2-cell stage. Starting from this stage, it was always found in nuclei of interphase blastomeres, where its "speckles" co-localized with the speckles containing high concentrations of snRNP polypeptides. SnRNP polypeptides recognized by anti-Sm and anti-RNP sera were in turn found in nuclei of all developmental stages. When embryos were treated with aphidicolin or cytochalasin D to arrest cell division, the 43B1N reacting protein was again localized in the pronuclei at 42 hr post-hCG, i.e., slightly later than the onset of transcriptional activity. These results suggest a progressive building up of nuclei during embryonic development, which could influence gene expression.

Competent mouse oocytes isolated from antral follicles exhibit different chromatin organization and follow different maturation dynamics.

After labelling DNA with the specific vital fluorophore Hoechst 33342, oocytes, isolated by puncture from antral follicles in adult mice, have two essentially different configurations of their nuclear fluorescence images. These have been called SN (where the nucleolus is surrounded by chromatin) and NSN (where the nucleolus is not surrounded by chromatin). Intermediate configurations are also found, although with a lower frequency. The proportion of each class is on the average equal and depends neither on the presence of cumulus cells nor on the age of the mouse. Electron microscopy confirms several ultrastructural differences between these two nuclear configurations, namely, the structure of the nucleolus, which is vacuolated in NSN-type and compact in SN-type oocytes. Using video-enhanced fluorescence microscopy at low level of excitation light, we could follow directly in vitro the meiotic maturation of both classes, without impairing their viability. We show that in germinal vessicle (GV) state, the chromatin does not change from one configuration into the other and that both classes are able to mature to metaphase II, although the maturation has slightly different characteristics.

Fertilization and ageing processes in non-divided human oocytes after GnRHa treatment: an analysis of individual oocytes.

Some human oocytes cultured together with spermatozoa for in-vitro fertilization (IVF) do not subsequently divide. The arrest of the fertilization process at different moments during development may provide information about the cause of fertilization failure. Oocytes which subsequently divide are transferred 48 h after insemination; when oocytes do not divide, ageing processes can be observed. Therefore these oocytes are interesting material in which to observe both fertilization and ageing. Our study concerns 72 undivided human oocytes 0, 48 or 72 h post-insemination. DNA of the oocyte and spermatozoa was visualized by the DNA fluorescent dye Hoechst 33342. Living oocytes were observed in toto by fluorescence and bright field microscopy which allowed nuclear and pronuclear membranes to be discerned. Oocytes were subsequently fixed and sectioned for bright field microscopy. Both techniques allowed parallel observations. Oocytes at various stages of fertilization are described: sperm penetration in both mature and immature oocytes, decondensation of sperm-heads, premature condensation of male chromatin, polyspermy and pronucleus formation. Typical ageing processes such as the centripetal migration of the metaphase II chromosomes, the formation of a restitution nucleus and the lagging of chromosomes within a metaphase spindle are observed. DNA fluorescence appears to be a quick, easy and valuable means to analyse fertilization and its failure.

Chromatin behaviour under influence of puromycin and 6-DMAP at different stages of mouse oocyte maturation.

Preovulatory mouse oocytes were cultured in vitro up to each subsequent stages of maturation: germinal vesicle (GV), germinal vesicle breakdown (GVBD), groups of not yet individualized bivalents, circular bivalents, late prometaphase I, metaphase I, anaphase I and telophase I. The stages were identified in living oocytes by fluorescence microscopy using Hoechst 33342 as a specific vital dye. Oocytes from each stage of development developed in vitro and ovulated metaphase II oocytes were subsequently cultured in the presence of puromycin or 6-dimethylaminopurine (6-DMAP), an inhibitor of protein phosphorylation. The effects on chromatin of these drugs were studied during and at the end of culture by fluorescence and electron microscopy. We found that puromycin and 6-DMAP stop meiosis when applied at all stages of oocyte maturation, except for metaphase II. Oocytes at this stage are activated by puromycin. Reaction of the oocytes to the two drugs is different at GV and at metaphase II. All of the other stages react to the drugs by chromatin compaction, which can be followed by chromatin decondensation to form a nucleus. Our results suggest that late prophase chromatin condensation, bivalent individualization and retention of their individuality, as well as individualization of monovalents from telophase and retention of their individuality at metaphase II, are dependent on protein phosphorylation. The events occurring between metaphase I and telophase I are independent of protein synthesis and phosphorylation. The events occurring between metaphase II and formation of the nucleus are independent of protein synthesis.