Systematic analysis of RhoGEF/GAP localizations uncovers regulators of mechanosensing and junction formation during epithelial cell division.

Cell proliferation is central to epithelial tissue development, repair, and homeostasis. During cell division, small RhoGTPases control both actomyosin dynamics and cell-cell junction remodeling to faithfully segregate the genome while maintaining tissue polarity and integrity. To decipher the mechanisms of RhoGTPase spatiotemporal regulation during epithelial cell division, we generated a transgenic fluorescently tagged library for the 48 Drosophila Rho guanine exchange factors (RhoGEFs) and GTPase-activating proteins (GAPs), and we systematically characterized their endogenous distributions by time-lapse microscopy. Therefore, we unveiled candidate regulators of the interplay between actomyosin and junctional dynamics during epithelial cell division. Building on these findings, we established that the conserved RhoGEF Cysts and RhoGEF4 play sequential and distinct roles to couple cytokinesis with de novo junction formation. During ring contraction, Cysts via Rho1 participates in the neighbor mechanosensing response, promoting daughter-daughter cell membrane juxtaposition in preparation to de novo junction formation. Subsequently and upon midbody formation, RhoGEF4 via Rac acts in the dividing cell to ensure the withdrawal of the neighboring cell membranes, thus controlling de novo junction length and cell-cell arrangements upon cytokinesis. Altogether, our findings delineate how the RhoGTPases Rho and Rac are locally and temporally activated during epithelial cytokinesis, highlighting the RhoGEF/GAP library as a key resource to understand the broad range of biological processes regulated by RhoGTPases.

Whole-Cell Photobleaching Reveals Time-Dependent Compartmentalization of Soluble Proteins by the Axon Initial Segment.

By limiting protein exchange between the soma and the axon, the axon initial segment (AIS) enables the segregation of specific proteins and hence the differentiation of the somatodendritic compartment and the axonal compartment. Electron microscopy and super-resolution fluorescence imaging have provided important insights in the ultrastructure of the AIS. Yet, the full extent of its filtering properties is not fully delineated. In particular, it is unclear whether and how the AIS opposes the free exchange of soluble proteins. Here we describe a robust framework to combine whole-cell photobleaching and retrospective high-resolution imaging in developing neurons. With this assay, we found that cytoplasmic soluble proteins that are not excluded from the axon upon expression over tens of hours exhibit a strong mobility reduction at the AIS - i.e., are indeed compartmentalized - when monitored over tens of minutes. This form of compartmentalization is developmentally regulated, requires intact F-actin and may be correlated with the composition and ultrastructure of the submembranous spectrin cytoskeleton. Using computational modeling, we provide evidence that both neuronal morphology and the AIS regulate this compartmentalization but act on distinct time scales, with the AIS having a more pronounced effect on fast exchanges. Our results thus suggest that the rate of protein accumulation in the soma may impact to what extent and over which timescales freely moving molecules can be segregated from the axon. This in turn has important implications for our understanding of compartment-specific signaling in neurons.

FTIR microscopy contribution for comprehension of degradation mechanisms in PLA-based implantable medical devices.

The integration and evolution of implantable medical devices made of bioresorbable polymers and used for temporary biomedical applications are crucial criteria in the success of a therapy and means of follow-up after implantation are needed. The objective of this work is to develop and evaluate a method based on microscopic Fourier Transform InfraRed spectroscopy (FTIR) mappings to monitor the degradation of such polymers on tissue explant sections, after implantation. This technique provided information on their location and on both their composition and crystallinity, which is directly linked to their state of degradation induced predominantly by chain scissions. An in vitro study was first performed on poly(L-lactic acid) (PLLA) meshes to validate the procedure and the assumption that changes observed on FTIR spectra are indeed a consequence of degradation. Then, mappings of in vivo degraded PLLA meshes were realized to follow up their degradation and to better visualize their degradation mechanisms. This work further warrants its translation to medical implants made of copolymers of lactic acid and to other polyesters.

[Biodegradable synthetic polymers for the design of implantable medical devices: the ligamentoplasty case]. / Polymères synthétiques dégradables pour la conception de dispositifs médicaux implantables - Le cas de la ligamentoplastie.

The sector of implantable medical devices is a growing sector of health products especially dynamic in the field of research. To improve the management of patients and to meet clinical requirements, researchers are developing new types of medical devices. They use different families of biomaterials presenting various chemical and physical characteristics in order for providing clinicians with health products optimized for biomedical applications. In this article, we aim to show how, starting from a family of biomaterials (degradable polymers), it is possible to design an implantable medical device for the therapeutic management of the failure of anterior cruciate ligament. The main steps leading to the design of a total ligament reinforcement are detailed. They range from the synthesis and characterization of degradable polymer to the shaping of the knitted implant, through the assessment of the study of the impact of sterilization on mechanical properties and checking cytocompatibility.

Rolled knitted scaffolds based on PLA-pluronic copolymers for anterior cruciate ligament reinforcement: A step by step conception.

The aim of this study was to prepare a new knitted scaffold from PLA-Pluronic block copolymers for anterior cruciate ligament reconstruction. The impact of sterilization methods (beta-ray and gamma-ray sterilization) on copolymers was first evaluated in order to take into account the possible damages due to the sterilization process. Beta-ray radiation did not significantly change mechanical properties in contrast to gamma-ray sterilization. It was shown that ACL cells proliferate onto these copolymers, demonstrating their cytocompatibility. Thirdly, in order to study the influence of shaping on mechanical properties, several shapes were created with copolymers yarns: braids, ropes and linear or rolled knitted scaffolds. The rolled knitted scaffold presented interesting mechanical characteristics, similar to native anterior cruciate ligament (ACL) with a 67 MPa Young's Modulus and a stress at failure of 22.5 MPa. These findings suggest that this three dimensional rolled knitted scaffold meet the mechanical properties of ligament tissues and could be suitable as a scaffold for ligament reconstruction. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 735-743, 2017.

PLA-poloxamer/poloxamine copolymers for ligament tissue engineering: sound macromolecular design for degradable scaffolds and MSC differentiation.

The treatment of anterior cruciate ligament (ACL) failures remains a current clinical challenge. The present study aims at providing suitable degradable scaffolds for ligament tissue engineering. First, we focus on the design and the evaluation of poly(lactide)/poloxamer or poly(lactide)/poloxamine multiblock copolymers selected and developed to have suitable degradation and mechanical properties to match ACL repair. In the second part, it is shown that the copolymers can be processed in the form of microfibers and scaffolds consisting of a combination of twisted/braided fibers to further modulate the mechanical properties and prepare scaffold prototypes suitable for ligament application. Finally, after assessment of their cytocompatibility, the polymer scaffolds are associated with mesenchymal stem cells (MSCs). MSC differentiation toward a ligament fibroblast phenotype is promoted by a dual stimulation including an inductive culture medium and cyclic mechanical loads. RT-qPCR analyses confirm the potential of our scaffolds and MSCs for ACL regeneration with upregulation of some differentiation markers including Scleraxis, Tenascin-C and Tenomodulin.

Investigation on the properties of linear PLA-poloxamer and star PLA-poloxamine copolymers for temporary biomedical applications.

The objective of this work was to develop and study new biodegradable thermoplastics with improved mechanical properties for potential use as temporary implantable biomaterials. Linear poloxamer and star-shaped poloxamine have been used as macroinitiators for the ring-opening polymerization (ROP) of lactide to yield high molecular weight PLA-based thermoplastic block copolymers. The influence of the nature of the macroinitiator, PLA crystallinity and initial molecular weight on the copolymers properties was investigated by performing a 7-week degradation test in PBS. The evaluation of water uptakes and molecular weights during the degradation pointed out an early hydrolytic degradation of the 100-kg∙mol(-1) copolymers compared to the 200-kg∙mol(-1) ones (molecular weight decrease of ca. 40% and 20%, respectively). A dramatic loss of tensile mechanical properties was also observed for the 100-kg∙mol(-1) copolymers, whereas the 200-kg∙mol(-1) copolymers showed stable or even slightly improved properties with Young's moduli around 500 MPa and yield strains around 3% to 4%. Finally, the cytocompatibility of the more stable 200 kg∙mol(-1) copolymers was confirmed by murine mesenchymal stem cells (MSCs) culture.

PLA-based biodegradable and tunable soft elastomers for biomedical applications.

Although desirable for biomedical applications, soft degradable elastomers having balanced amphiphilic behaviour are rarely described in the literature. Indeed, mainly highly hydrophobic elastomers or very hydrophilic elastomers with hydrogel behaviours are found. In this work, we developed thermoset degradable elastomers based on the photo-cross-linking of poly(lactide)-poly(ethylene glycol)-poly(lactide) (PLA-PEG-PLA) triblock prepolymers. The originality of the proposed elastomers comes from the careful choice of the prepolymer amphiphilicity and from the possible modulation of their mechanical properties and degradation rates provided by cross-linkers of different nature. This is illustrated with the hydrophobic and rigid 2,4,6-triallyloxy-1,3,5-triazine compared to the hydrophilic and soft pentaerythritol triallyl ether. Thermal properties, mechanical properties, swelling behaviours, degradation rates and cytocompatibility have been evaluated. Results show that it is possible to generate a family of degradable elastomers covering a broad range of properties from a single biocompatible and biodegradable prepolymer.