Biology subjects

PICART, C.

Publications and source records attributed to PICART, C..

6 recordsLinked to original sources

bioRxiv2023

Development of an automated high-content immuno-fluorescence assay of pSmads quantification: proof-of-concept with drugs inhibiting the BMP/TGFbeta pathways

Bone morphogenetic proteins (BMPs) and transforming growth factors (TGF-{beta}) are members of the transforming growth factors superfamily, known for their role in several physiological and pathological processes. These factors are known to bind in vivo to BMP and TGF-{beta} receptors respectively, which induces the phosphorylation of the Smad (pSmad) transcription factors. This pathway is generally studied with western blot and luciferase bioluminescence assay, which present some limitations. In our work, we developed and optimized a high-content immunofluorescence assay to study the pSmad pathway on glass as well as on biomaterials by overcoming the technical challenges raised by image acquisition and analysis. Furthermore, with this assay, we present here a proof-of concept for drug testing on glass and on biomimetic films using drug inhibitors of the BMP receptor and of the TGF-{beta} receptors. Altogether, our results open perspectives for future drug testing on biomimetic films that present various growth factors and extracellular matrix proteins or peptides.

bioengineering↗

bioRxiv2022

3D-printed polymeric scaffolds with optimized architecture to repair a sheep metatarsal critical-size bone defect

The reconstruction of critical-size bone defects in long bones remains a challenge for clinicians. We developed a new bioactive medical device for long bone repair by combining a 3D-printed architectured cylindrical scaffold made of clinical-grade polylactic acid (PLA) with a polyelectrolyte film coating delivering the osteogenic bone morphogenetic protein 2 (BMP-2). This film-coated scaffold was used to repair a sheep metatarsal 25-mm long critical-size bone defect. In vitro and in vivo biocompatibility of the film-coated PLA material were proved according to ISO standards. Scaffold geometry was found to influence BMP-2 incorporation. Bone regeneration was followed using X-ray scans, {micro}CT scans, and histology. We showed that scaffold internal geometry, notably pore shape, influenced bone regeneration, which was homogenous longitudinally. Scaffolds with cubic pores of [~]870 {micro}m and a low BMP-2 dose of [~]120 {micro}g/cm3 induced the best bone regeneration without any adverse effects. The visual score given by clinicians during animal follow-up was found to be an easy way to predict bone regeneration. This work opens perspectives for a clinical application in personalized bone regeneration.

bioengineering↗

bioRxiv2021

Differential bioactivity of four BMP-family members as function of biomaterial stiffness

Whereas soft biomaterial is not able to induce cell spreading, BMP-2 presented by a soft film has been described to be sufficient to trigger cell spreading, migration and downstream BMP-2 signaling. Based on thin polyelectrolyte films of controlled stiffness, we investigated whether the presentation of four BMP members (2, 4, 7, 9) in a matrix-bound manner may differentially impact cell adhesion and bone differentiation of skeletal progenitors. We performed high content and automated screening of cellular responses, including cell number, cell spreading area, SMAD phosphorylation and alkaline phosphatase activity. The basolateral presentation of the different BMPs allowed us to discriminate the specificity of cellular response and the role of BMP receptors type I, type II, as well as three {beta} integrins, in a BMP type and stiffness-dependent manner.

cell biology↗

bioRxiv2020

3D printed scaffold combined to 2D osteoinductive coatings to repair a critical-size mandibular bone defect

the reconstruction of large bone defects (12 cm3) remains a challenge for clinicians. We developed a new critical-size mandibular bone defect model on a mini-pig, close to human clinical issues. We analyzed the bone reconstruction obtained by a 3D printed scaffold made of clinical-grade PLA, coated with a polyelectrolyte film delivering an osteogenic bioactive molecule (BMP-2). We compared the results (CT-scan, CT, histology) to the gold standard solution, bone autograft. We demonstrated that the dose of BMP-2 delivered from the scaffold significantly influenced the amount of regenerated bone and the repair kinetics, with a clear BMP-2 dose-dependence. Bone was homogeneously formed inside the scaffold without ectopic bone formation. The bone repair was as good as for the bone autograft. The BMP-2 doses applied in our study were reduced 20 to 75-fold compared to the commercial collagen sponges used in the current clinical applications, without any adverse effects. 3D printed PLA scaffolds loaded with reduced doses of BMP-2 can be a safe and simple solution for large bone defects faced in the clinic.

bioengineering↗

bioRxiv2020

High throughput measurements of BMP/BMP receptors interactions using bio-layer interferometry

Bone morphogenetic proteins (BMP) are an important family of growth factors playing a role in a large number of physiological and pathological processes, including bone homeostasis, tissue regeneration and cancers. In vivo, BMPs bind successively to both BMP receptors (BMPR) of type I and type II, and a promiscuity has been reported. In this study, we used bio-layer interferometry to perform parallel real-time biosensing and to deduce the kinetic parameters (ka, kd) and the equilibrium constant (KD) for a large range of BMPs/BMPR combinations in similar experimental conditions. We selected four members of the BMP family (BMP-2, 4, 7, 9) known for their physiological relevance and studied their interactions with five type-I BMP receptors (ALK1, 2, 3, 5, 6) and three type-II BMP receptors (BMPR-II, ACTR-IIA, ACTR-IIB). We reveal that BMP-2 and BMP-4 behave differently, especially regarding their kinetic interactions and affinities with the type-II BMPR. We found that BMP-7 has a higher affinity for ACTR-IIA and a tenfold lower affinity with the type-I receptors. While BMP-9 has a high and similar affinity for all type-II receptors, it can interact with ALK5 and ALK2, in addition to ALK1. Interestingly, we also found that all BMPs can interact with ALK5. The interaction between BMPs and both type-I and type II receptors immobilized on the same surface did not reveal further cooperativity. Our work provides a synthetic view of the interactions of these BMPs with their receptors and paves the way for future studies on their cell-type and receptor specific signaling pathways.

biophysics↗

bioRxiv2016

Self assembly of HIV-1 Gag protein on lipid membranes generates PI(4,5)P2/Cholesterolnanoclusters.

The self-assembly of HIV-1 Gag polyprotein at the inner leaflet of the cell host plasma membrane is the key orchestrator of virus assembly. The binding between Gag and the plasma membrane is mediated by specific interaction of the Gag matrix domain and the PI(4,5)P2 lipid (PIP2). It is unknown whether this interaction could lead to local reorganization of the plasma membrane lipids. In this study, using model membranes, we examined the ability of Gag to segregate specific lipids upon self-assembly. We show for the first time that Gag self-assembly is responsible for the formation of PIP2 lipid nanoclusters, enriched in cholesterol but not in sphingomyelin. We also show that Gag mainly partition into liquid-disordered domains of these lipid membranes. Our work strongly suggests that, instead of targeting pre-existing plasma membrane lipid domains, Gag is more prone to generate PIP2/Cholesterol lipid nanodomains at the inner leaflet of the plasma membrane during early events of virus assembly.

biophysics↗