Gabriela Rodrigues

In vitro and in vivo evaluation of electrospun nanofibers of PCL, chitosan and gelatin: a comparative study.

Many polymers have been investigated with respect to their use in skin tissue engineering. However, directly comparable data on the role played by different polymers in assisting skin wound healing requires their in vitro and in vivo evaluation under the same conditions. Therefore, we performed a study in order to compare the performance of electrospun nanofiber mats from three different polymers concerning cell-scaffold interaction and wound healing promotion. A polyester (polycaprolactone, PCL), a protein (gelatin from cold water fish skin, GEL) and a polysaccharide (chitosan, CS) were the polymers chosen. Gelatin nanofibers were crosslinked with glutaraldehyde vapor. The scaffolds were characterized physico-chemically, in vitro by seeding with human fetal fibroblasts, HFFF2, and used in vivo as skin substitutes in a rat wound model with total skin removal. In vitro tests revealed that cells adhered and proliferated in all scaffolds. However, cells deep into the scaffold were only observed in the PCL and CS scaffolds. In in vivo tests CS scaffolds had the highest impact on the healing process by decreasing the extent of wound contraction and enhancing the production of a neodermis and re-epithelialization of the wound.

Redefining the role of ectoderm in somitogenesis: a player in the formation of the fibronectin matrix of presomitic mesoderm.

The absence of ectoderm impairs somite formation in cultured presomitic mesoderm (PSM) explants, suggesting that an ectoderm-derived signal is essential for somitogenesis. Here we show in chick that the standard enzymatic treatments used for explant isolation destroy the fibronectin matrix surrounding the anterior PSM, which fails to form somites when cultured for 6 hours. By contrast, explants isolated with collagenase retain their fibronectin matrix and form somites under identical culture conditions. The additional presence of ectoderm enhances somite formation, whereas endoderm has no effect. Furthermore, we show that pancreatin-isolated PSM explants cultured in fibronectin-supplemented medium, form significantly more somites than control explants. Interestingly, ectoderm is the major producer of fibronectin (Fn1) transcripts, whereas all but the anterior-most region of the PSM expresses the fibronectin assembly receptor, integrinα 5 (Itga5). We thus propose that the ectoderm-derived fibronectin is assembled by mesodermal α5β1 integrin on the surface of the PSM. Finally, we demonstrate that inhibition of fibronectin fibrillogenesis in explants with ectoderm abrogates somitogenesis. We conclude that a fibronectin matrix is essential for morphological somite formation and that a major, previously unrecognised role of ectoderm in somitogenesis is the synthesis of fibronectin.

Dynamic 3D Cell Rearrangements Guided by a Fibronectin Matrix Underlie Somitogenesis

Somites are transient segments formed in a rostro-caudal progression during vertebrate development. In chick embryos, segmentation of a new pair of somites occurs every 90 minutes and involves a mesenchyme-to-epithelium transition of cells from the presomitic mesoderm. Little is known about the cellular rearrangements involved, and, although it is known that the fibronectin extracellular matrix is required, its actual role remains elusive. Using 3D and 4D imaging of somite formation we discovered that somitogenesis consists of a complex choreography of individual cell movements. Epithelialization starts medially with the formation of a transient epithelium of cuboidal cells, followed by cell elongation and reorganization into a pseudostratified epithelium of spindle-shaped epitheloid cells. Mesenchymal cells are then recruited to this medial epithelium through accretion, a phenomenon that spreads to all sides, except the lateral side of the forming somite, which epithelializes by cell elongation and intercalation. Surprisingly, an important contribution to the somite epithelium also comes from the continuous egression of mesenchymal cells from the core into the epithelium via its apical side. Inhibition of fibronectin matrix assembly first slows down the rate, and then halts somite formation, without affecting pseudopodial activity or cell body movements. Rather, cell elongation, centripetal alignment, N-cadherin polarization and egression are impaired, showing that the fibronectin matrix plays a role in polarizing and guiding the exploratory behavior of somitic cells. To our knowledge, this is the first 4D in vivo recording of a full mesenchyme-to-epithelium transition. This approach brought new insights into this event and highlighted the importance of the extracellular matrix as a guiding cue during morphogenesis.

Fibronectin promotes migration, alignment and fusion in an in vitro myoblast cell model

Myogenesis is a complex process in which committed myogenic cells differentiate and fuse into myotubes that mature into the muscle fibres of adult organisms. This process is initiated by a cascade of myogenic regulatory factors expressed upon entry of the cells into the myogenic differentiation programme. However, external signals such as those provided by the extracellular matrix (ECM) are also important in regulating muscle differentiation and morphogenesis. In the present work, we have addressed the role of various ECM substrata on C2C12 myoblast behaviour in vitro. Cells grown on fibronectin align and fuse earlier than cells on laminin or gelatine. Live imaging of C2C12 myoblasts on fibronectin versus gelatine has revealed that fibronectin promotes a directional collective migratory behaviour favouring cell-cell alignment and fusion. We further demonstrate that this effect of fibronectin is mediated by RGD-binding integrins expressed on myoblasts, that N-cadherin contributes to this behaviour, and that it does not involve enhanced myogenic differentiation. Therefore, we suggest that the collective migration and alignment of cells seen on fibronectin leads to a more predictable movement and a positioning that facilitates subsequent fusion of myoblasts. This study highlights the importance of addressing the role of fibronectin, an abundant component of the interstitial ECM during embryogenesis and tissue repair, in the context of myogenesis and muscle regeneration.

In vitro evaluation of crosslinked electrospun fish gelatin scaffolds.

Gelatin from cold water fish skin was electrospun, crosslinked and investigated as a substrate for the adhesion and proliferation of cells. Gelatin was first dissolved in either water or concentrated acetic acid and both solutions were successfully electrospun. Cross-linking was achieved via three different routes: glutaraldehyde vapor, genipin and dehydrothermal treatment. Solutions properties (surface tension, electrical conductivity and viscosity) and scaffolds properties (chemical bonds, weight loss and fiber diameters) were measured. Cellular viability was analyzed culturing 3T3 fibroblasts plated on the scaffolds and grown up to 7 days. The cells were fixed and observed with SEM or stained for DNA and F-actin and observed with confocal microscopy. In all scaffolds, the cells attached and spread with varying degrees. The evaluation of cell viability showed proliferation of cells until confluence in scaffolds crosslinked by glutaraldehyde and genipin; however the rate of growth in genipin crosslinked scaffolds was slow, recovering only by day five. The results using the dehydrothermal treatment were the less satisfactory. Our results show that glutaraldehyde treated fish gelatin is the most suitable substrate, of the three studied, for fibroblast adhesion and proliferation.

Evaluation of nanofibrous scaffolds obtained from blends of chitosan, gelatin and polycaprolactone for skin tissue engineering

Polymer blending is a strategy commonly used to obtain hybrid materials possessing properties better than those of the individual constituents regarding their use in scaffolds for Tissue Engineering. In the present work, the scaffolds produced by electrospinning solutions of polymeric blends obtained using a polyester (polycaprolactone, PCL), a polysaccharide (chitosan, CS) and a protein (gelatin extracted from cold water fish skin, GEL), were investigated. Solutions conductivity, shear viscosity and surface tension were determined. GEL-containing scaffolds were crosslinked with vapour phase glutaraldehyde (GTA). The scaffolds were characterized physico-chemically regarding fibre morphology, porosity, water contact angle, mechanical properties, chemical bonds and fibre and dimensional stability upon immersion in water and cell culture medium. The scaffolds were further tested in vitro for cell adhesion, growth and morphology of human foetal fibroblasts (cell line HFFF2). Results show that the nanofibrous scaffolds are hydrophilic and display the typical porosity of non-woven fibre mats. The CS/PCL and CS/PCL/GEL scaffolds have the highest elastic modulus (48MPa). Dimensional stability is best for the CS/PCL/GEL scaffolds. FTIR spectra confirm the occurrence of cross-linking reactions of GTA with both GEL and CS. Cell adhesion ratio ranked from excellent (close to 100%) to satisfactory (around 50%) in the order PCL/GEL>CS/GEL>CS/PCL/GEL>CS/PCL. Cell populations show an extended lag phase in comparison with the controls but cell proliferation occurs on all scaffolds until confluence is reached. In conclusion, all scaffolds studied possess characteristics that enable them to be used in skin tissue engineering but the CS/PCL/GEL scaffolds have better physical properties whereas the PCL/GEL scaffolds support a higher cell adhesion.

Chicken primordial germ cells use the anterior vitelline veins to enter the embryonic circulation

Summary During gastrulation, chicken primordial germ cells (PGCs) are present in an extraembryonic region of the embryo from where they migrate towards the genital ridges. This is also observed in mammals, but in chicken the vehicle used by the migratory PGCs is the vascular system. We have analysed the migratory pathway of chicken PGCs, focusing on the period of transition from the extraembryonic region to the intraembryonic vascular system. Our findings show that at Hamburger and Hamilton developmental stage HH12–HH14 the majority of PGCs concentrate axially in the sinus terminalis and favour transport axially via the anterior vitelline veins into the embryonic circulation. Moreover, directly blocking the blood flow through the anterior vitelline veins resulted in an accumulation of PGCs in the anterior region and a decreased number of PGCs in the genital ridges. We further confirmed the key role for the anterior vitelline veins in the correct migration of PGCs using an ex ovo culture method that resulted in defective morphogenetic development of the anterior vitelline veins. We propose a novel model for the migratory pathway of chicken PGCs whereby the anterior vitelline veins play a central role at the extraembryonic and embryonic interface. The chicken model of PGC migration through the vasculature may be a powerful tool to study the process of homing (inflammation and metastasis) due to the striking similarities in regulatory signaling pathways (SDF1–CXCR4) and the transient role of the vasculature.

Extracellular matrix remodeling accompanies axial muscle development and morphogenesis in the mouse.

Background: Skeletal myogenesis is extensively influenced by the surrounding environment. However, how the extracellular matrix (ECM) affects morphogenesis of muscles is not well understood. Results: We mapped the three‐dimensional (3D) organization of fibronectin, tenascin, and laminin by immunofluorescence during early epaxial myogenesis in mouse embryos. We define four stages of dermomyotome/myotome development and reveal the 3D organization of myogenic cells within their ECM during those stages. Fibronectin is abundant in all interstitial tissues, while tenascin is restricted to intersegmental borders. Bundles of fibronectin and tenascin also penetrate into the myotome, possibly promoting myocyte alignment. A laminin matrix delineates the dermomyotome and myotome and undergoes dynamic changes, correlating with key developmental events. Conclusion: Our observations cast new light on how myotomal cells interact with their environment and suggest that, as the segmented myotomes transform into the epaxial muscle masses, the laminin matrix disassembles and myocytes use the abundant fibronectin matrix to reach their final organization. Developmental Dynamics 241:350–364, 2012.

TACC3 Is Important for Correct Progression of Meiosis in Bovine Oocytes

Transforming acidic coiled-coil (TACC) proteins are key players during mitosis via stabilization of the spindle. The roles of TACCs during meiosis are however less clear. We used bovine oocytes to study the expression and function of TACC3 during meiosis. TACC3 mRNA was detected in bovine oocytes during meiosis using qRT-PCR, and while it was also expressed in cleavage stage embryos, its expression was down-regulated at the morula and blastocyst stages. Immunofluorescence was used to demonstrate that TACC3 co-localized with tubulin in the metaphase I and II spindles. However, TACC3 was not detected at anaphase or telophase of the first meiotic division. Aurora A, which is known to phosphorylate and activate TACC3 in mitotic cells, showed a similar pattern of gene expression to that of TACC3 in meiotic oocytes and preimplantation embryos. Aurora A protein was however only very transiently associated to the meiotic spindle. Pharmaceutical inhibition of Aurora A activity inhibited TACC3 phosphorylation but did not prevent TACC3 appearance in the spindle. Inhibiting Aurora A activity did however lead to abnormal meiotic spindle formation and impaired maturation of bovine oocytes. Similar results were obtained by knock-down of TACC3 expression using siRNA injection. These results suggest that TACC3 is important for stabilizing the meiotic spindle, but phosphorylation of TACC3 by Aurora A is not required for its recruitment to the meiotic spindle although phosphorylation of TACC3 by other kinases cannot be excluded.

Chitosan-Based Matrices Prepared by Gamma Irradiation for Tissue Regeneration: Structural Properties vs. Preparation Method

In the last decade, new generations of biopolymer-based materials have attracted attention, aiming its application as scaffolds for tissue engineering. These engineered three-dimensional scaffolds are designed to improve or replace damaged, missing, or otherwise compromised tissues or organs. Despite the number of promising methods that can be used to generate 3D cell-instructive matrices, the innovative nature of the present work relies on the application of ionizing radiation technology to form and modify surfaces and matrices with advantage over more conventional technologies (room temperature reaction, absence of harmful initiators or solvents, high penetration through the bulk materials, etc.), and the possibility of preparation and sterilization in one single step. The current chapter summarizes the work done by the authors in the gamma radiation processing of biocompatible and biodegradable chitosan-based matrices for skin regeneration. Particular attention is given to the correlation between the different preparation conditions and the final polymeric matrices’ properties. We therefore expect to demonstrate that instructive matrices produced and improved by radiation technology bring to the field of skin regenerative medicine a supplemental advantage over more conservative techniques.