Biology subjects

Le Visage, C.

Publications and source records attributed to Le Visage, C..

5 recordsLinked to original sources

bioRxiv2026

Advanced microfluidic strategy for In-Bead MSC spheroid formation and co-encapsulation of necrosis inhibitor-loaded nanoparticles

Mesenchymal stem/stromal cells (MSCs) are key players in regenerative medicine due to their immunomodulatory properties and ability to promote tissue repair. However, their therapeutic efficacy is often limited by rapid clearance following transplantation. MSC spheroids have shown enhanced functional properties, and we hypothesize that encapsulating them within hydrogel microbeads could offer additional protection and improve their viability. In this study, we developed a novel droplet-based microfluidic protocol for human MSCs derived from the apical papilla (SCAP) encapsulation and In-Bead spheroid formation within alginate microbeads. Optimization of the protocol allowed the formation of MSC spheroids in alginate droplets overnight (In-Bead), before alginate cross-linking and retrieval of alginate beads loaded with MSC spheroids. SCAP were successfully encapsulated within 275 {micro}m alginate microbeads, forming spheroids of approximately 80 {micro}m in diameter. Encapsulated SCAP spheroids retained their immunomodulatory properties. The process was further optimized by incorporating nanomedicines into the alginate solution before the formation of droplets and then spheroids, forming thus hybrid beads (Sph.Beads/NP). Nanomedicines were loaded with NecroX-5, a necrosis inhibitor, to improve SCAP viability further. Live/Dead assays indicated a protective effect of the nanomedicines, supporting the potential of this system for advanced cell delivery in regenerative applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/732109v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1625a4eorg.highwire.dtl.DTLVardef@16c6faorg.highwire.dtl.DTLVardef@161819aorg.highwire.dtl.DTLVardef@83c0d7_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical abstractA combination strategy enhancing MSC viability through spheroid formation, microencapsulation, and nanomedicine association achieved by microfluidic encapsulation with In-Bead spheroid formation. Created with BioRender

bioengineering↗

bioRxiv2025

A degradable nanofibrous scaffold of poly(ε-Caprolactone-co-Lactide) for annulus fibrosus repair

The intervertebral disc (IVD) is a key contributor to the spines biomechanical functions. It consists of a gelatinous core (nucleus pulposus, NP) surrounded by a fibrous ring (annulus fibrosus, AF). Accumulation of microcracks and tears in the AF can lead to NP herniation outside the disc space, compressing the nerve roots and causing pain. Herniation is a leading cause of low back pain and represents a major socioeconomic burden, with no effective regenerative treatment currently available. Previously, we demonstrated the potential of a poly({varepsilon}-caprolactone) (PCL)-based implant for the closure of an annulus fibrosus (AF) defect. However, the slow in vivo degradation of PCL may hinder timely AF regeneration. Here, we hypothesized that accelerating PCL degradation kinetics by incorporating a lactide component, known for its rapid degradation, could enhance AF repair. To that aim, PCLA polymers of {varepsilon}-caprolactone and lactide were synthesised as a copolymer (C-CL90%LA10%) or a blend (B-PCL80%PLA20%). These polymers were electrospun into aligned nanofibrous sheets, which were then assembled into a biomimetic multi-lamellar 3D implant. Cytocompatibility was assessed in vitro using ovine AF cells and ex vivo using a bovine tail disc model. In vitro, PCLA sheets promoted ovine AF cell alignment, proliferation, and expression of type I and II collagen. Ex vivo, the multi-lamellar implant remained in place within a full-thickness (4 mm) annular defect in bovine tail discs for 4 weeks, with initial cell infiltration. Degradation and regenerative potential were then evaluated in an ovine lumbar full-thickness annular defect model at 1 month (n=4) and 6 months (n=10) after implantation. Histological and immunohistochemical analyses revealed substantial cellular infiltration and neo-tissue ingrowth within implant layers, despite implant displacement in some cases. At 1 month, all implants retained their multi-lamellar structure with no visible degradation. By 6 months, the copolymer implants exhibited marked degradation, whereas PCL and blend implants remained structurally intact. Furthermore, ex vivo biomechanical testing revealed comparable flexibility to intact but much lower than non-implanted controls in axial rotation. This study confirms the accelerated degradation kinetics of PCLA copolymers within the disc microenvironment and demonstrates that the multi-lamellar PCLA implant can guide AF tissue repair. Further optimization is needed to enhance implant retention and ensure long-term functional integration.

bioengineering↗

bioRxiv2025

Transcriptomic and functional comparison of cells isolated from healthy and degenerated ovine intervertebral discs

Structured abstractO_ST_ABSBackgroundC_ST_ABSIntervertebral disc degeneration (IVDD) is a leading cause of chronic low back pain and disability. Understanding the cellular and molecular mechanisms underlying disc degeneration is crucial for developing effective therapies. Sheep have emerged as a promising large-animal model for IVDD research due to their similarities with humans. They exhibit resembling spine anatomy and biomechanics, and they develop spontaneous age-associated degeneration of the disc. However, the specific cellular alterations occurring in annulus fibrosus (AF) and nucleus pulposus (NP) ovine cells during degeneration remain poorly characterized. In vitro, the benefits of using cells from aged sheep over young ones to mimic degenerative processes remain to be tested. MethodsAF and NP cells from young and aged sheep were analysed using bulk RNA sequencing, with a focus on two hallmarks of IVDD: cellular senescence and metabolic alterations. Functional assays completed this focus by assessing cells response under basal conditions and after pro-degenerative stimuli (IL-1{beta}, senescence induction). In addition, bulk transcriptomic data were deconvoluted using a reference single-cell RNA-seq dataset from healthy and degenerated human discs, and gene co-expression modules were compared across species. ResultsMRI and histological analyses revealed homogeneous mild degeneration across all lumbar discs in aged sheep, while lamb discs were uniformly healthy. Cells transcriptomic profiling identified robust age- and tissue-specific signatures, with aged NP and AF cells showing upregulation of inflammatory mediators, ECM-remodelling enzymes, and senescence-associated pathways. Cross-species analysis revealed shared transcriptional modules between aged sheep cells and human degenerated disc cells, supporting the translational relevance of the ovine model. Remarkably, young and aged cells shared a similar functional behaviour when exposed to stress-related stimuli. ConclusionsThis work confirms the compatibility of sheep cells with in vitro testing and their relevance to model human IVDD. Cross-validation with human single-cell data further highlights common pathogenic pathways, reinforcing the translational potential of the model. However, no added benefits were found in using older animals compared to younger ones as cell sources in functional assays. HighlightsO_LITranscriptomic profiling of AF and NP cells from young and aged sheep C_LIO_LIAged cells show inflammatory, ECM-remodelling and senescence signatures C_LIO_LIDeconvolution with human scRNA-seq links aged ovine and degenerated discs C_LIO_LISheep cells retain in vitro responsiveness to pro-degenerative stimuli C_LIO_LISupports the ovine model as a translational tool for IVDD research C_LI

cell biology↗

bioRxiv2025

BIOFABRICATION OF AN OVINE INTERVERTEBRAL DISC MODEL BY COMBINING A POLYCAPROLACTONE FRAME WITH A BIOPRINTED ALGINATE HYDROGEL

The intervertebral disc (IVD) primarily comprises an outer ring of collagen fibers (annulus fibrosus, AF), which encases a soft, gelatinous core (nucleus pulposus, NP). Existing in vitro models have failed to integrate these two tissues effectively or accurately replicate their intricate organization. By combining two biofabrication techniques, we developed a novel 3D in vitro model that closely mimics the organization of an ovine IVD. Our approach employs a polycaprolactone (PCL) frame produced via melt electrowriting to recreate the multilamellar architecture of the annulus fibrosus. Ovine primary cells, encapsulated in a photocrosslinkable alginate hydrogel, were precisely extruded within the multilamellar structure, thereby mimicking the native shape and size of an ovine disc. The bioink containing the NP cells was deposited at the center of the construct, while the bioink with the AF cells was strategically layered in between the lamellae of the PCL frame. Photocrosslinking was optimized to match the native stiffness of the disc. The constructs were maintained in culture for 28 days, during which we thoroughly assessed reproducibility, stability, and cell viability and phenotype. The results unequivocally demonstrated that the PCL frame effectively guided the alignment and proliferation of AF cells, while the alginate hydrogel preserved NP cell phenotype. This model successfully replicates the organization of the IVD, providing a promising platform for advancing our understanding of disc biology and driving the development of novel therapeutic strategies.

bioengineering↗

bioRxiv2023

Microencapsulation of mesenchymal stromal cells in covalent alginate hydrogels for cell therapy

Osteoarthritis (OA) is the most common inflammatory joint disease and currently lacks an effective curative treatment. Intra-articular injection of mesenchymal stromal cells (MSCs) has gained attention as a relevant therapeutic approach for OA treatment due to the MSCs ability to secrete anti-inflammatory and immunomodulatory factors. Given their limited viability post- intraarticular injection and the potential leakage of cells out of the injection site, encapsulating MSCs in hydrogels is considered a promising strategy to protect them and provide a suitable 3D microenvironment to support their biological activities. Calcium-cross-linked alginate hydrogels are commonly used for MSC encapsulation, but their long-term in vivo stability remains uncertain. On the other hand, alginate cross-linking by the strain-promoted azide- alkyne cycloaddition (SPAAC) reaction would create a network unaffected by an ionic environment. Hence, this study aimed to develop an alginate-based hydrogel cross-linked via stable and cytocompatible covalent bonds for cell encapsulation. We established for the first time the formation of covalent alginate hydrogels between two SPAAC precursors, namely alginate-BCN and alginate-N3. These hydrogels exhibited in vitro stability and enabled the diffusion of molecules of interest. We then generated alginate-based SPAAC microgels of 170 m in mean diameter, suitable for intra-articular injection. We next encapsulated human adipose MSCs (hASCs) in these alginate-based SPAAC microgels and confirmed their cytocompatibility, with over 90 % of cells remaining viable after 14 days in culture. Finally, the microencapsulated hASCs maintained their biological properties and were able to secrete anti-inflammatory factors (IDO, PGE2, and HGF) when exposed to pro-inflammatory cytokines (TNF- and IFN-{gamma}). In the end, human-activated lymphocytes were cultured in contact with microencapsulated hASCs, and CD3+ T cell proliferation was quantified by flow cytometry. We demonstrated that the encapsulation process did not impair the hASC immunomodulatory activity. Overall, our findings show the potential of alginate-based SPAAC hydrogels for microencapsulating hASCs for cell therapy.

bioengineering↗