Biofabricating the next generation of a whole intervertebral disc model

The intervertebral disc (IVD) is composed of a fibrous annulus fibrosus (AF), a gelatinous nucleus pulposus (NP), and cartilaginous endplates at the interface with the vertebrae, enabling nutrient diffusion into the avascular IVD. IVD degeneration is associated with low back pain, which affects more than 80% of adults during their lifetime and results in significant disability and socioeconomic burden.

Patients who have exhausted conservative treatments (analgesics and physiotherapy) are left with no alternative but invasive and costly surgical interventions. Bioinspired regenerative strategies have recently attracted increasing attention, and preclinical animal models have been developed to assess their efficacy. However, ethical guidelines and concerns regarding animal welfare have led to the adoption of the 3Rs principle (Replacement, Reduction, Refinement), highlighting the need for alternatives to animal experimentation.

Conventional two-dimensional cell cultures fail to recapitulate the complex three-dimensional structure of the IVD and do not accurately reflect in vivo responses. Therefore, advanced 3D preclinical models are required. We have previously developed a partial model of a healthy IVD that reproduces the spatial organization of the nucleus pulposus and partially that of the annulus fibrosus.

The aim of this doctoral project is to further develop this three-dimensional IVD model by incorporating the third compartment, namely the cartilaginous endplate (CEP), and by improving the physical and mechanical properties of the annulus fibrosus (AF). To achieve this, a biofabrication strategy using natural polymer-based bioinks will be employed. This model will subsequently be used to study IVD degeneration by incorporating relevant biological and biomechanical components of the pathology.

The first part of the project will focus on the development of bioinks (biopolymers + cells) intended to mimic the annulus fibrosus and cartilaginous endplates using 3D printing technologies. A systematic study will be conducted to generate cell-laden collagen-based hydrogels with tunable mechanical, physical, and biochemical properties. These formulations must support the survival of cells such as fibroblasts and mesenchymal stem cells. Initially, collagen concentration will be optimized to ensure adequate cell viability, appropriate morphology, and hydrogel stability (i.e., absence of contraction). Subsequently, hyaluronic acid will be incorporated to enhance the hydration capacity of the bioinks and better mimic the biochemical properties of the native IVD. Hydrogels will be characterized using rheology, scanning electron microscopy (SEM), and differential scanning calorimetry (DSC). Cell viability will be assessed using live/dead assays, and cell morphology will be analyzed by confocal microscopy following phalloidin staining.

The second part of the thesis will focus on the fabrication of biomaterials mimicking the CEP and AF using the selected bioinks. The CEP is a thin tissue (~1 mm) composed of a non-mineralized region (adjacent to the AF) and a mineralized region (adjacent to the subchondral bone). The proposed strategy involves printing a dense, cell-laden collagen layer to achieve a stiffness of approximately 10 kPa, alongside a similar layer incorporating hydroxyapatite nanoparticles. Printing parameters will be adjusted to modulate biomaterial porosity. A porous structure will replicate physiological CEP characteristics, whereas a low-porosity structure will mimic pathological conditions associated with IVD degeneration. Mechanical properties will be assessed using tensile testing, and structural features will be analyzed using SEM. Encapsulated mesenchymal stem cells will be differentiated in situ into chondrocyte-like cells. Cellular phenotype will be evaluated using quantitative PCR (qPCR), confocal microscopy, and flow cytometry, with particular attention to chondrogenic markers such as collagen type II, aggrecan, and type X collagen.

To mimic the annulus fibrosus, an elastic, deformable, and anisotropic structure composed of polycaprolactone (PCL) will be employed. The bioink composed of collagen,  hyaluronan,   fibroblasts or AF cells will be printed between PCL layers produced by melt electrowriting. Polysaccharide content will be optimized to ensure formulation stability and promote fibroblast spreading in the scaffold. Cellular phenotype will be assessed using qPCR and confocal microscopy.

The final part of the project will focus on assembling the biomaterials mimicking the different regions of the intervertebral disc to obtain a cohesive model suitable for studying IVD degeneration. The biomaterials developed during this doctoral project will be combined with a nucleus pulposus-mimicking hydrogel previously developed by the AO Foundation.

This project is a bilateral collaboration between France and Switzerland funded by the French National Research Agency (ANR). It involves the RMeS laboratory at the University of Nantes (France), the LCMCP (France), and the AO Foundation in Davos (Switzerland).

L'équipe « Matériaux et Biologie » du Laboratoire de Chimie de la Matière Condensée (LCMCP), animée par Francisco M. Fernandes, rassemble des chimistes, biologistes et physiciens autour de thématiques visant à développer des matériaux « vivants », en nous appuyant sur une meilleure compréhension des interactions cellules-matériaux. Notre stratégie repose sur l’intégration de fonctionnalités spécifiques de la biologie (auto-organisation, reconnaissance moléculaire, activité enzymatique, métabolisme cellulaire,..) au sein de matériaux et nanomatériaux polymériques, hybrides ou inorganiques, pour l’élaboration de biomatériaux biomimétiques, le développement de nouveaux dispositifs biotechnologiques 
 

Nos champs d’application dans le domaine médica s’appuient sur la mise en commun de ces champs d’expertise  et sur un large réseau de collaborations nationales et internationales pluridisciplinaires. Citons en particulier: 

– Cornées artificielles biomimétiques
– Biomatériaux mixtes et nanocomposites à base de collagène pour la réparation et la régénération tissulaire
– Modèles 3D du disque intervertébral
– Fabrication additive de biomatériaux pour la réparation cardiaque
– Cryo-procédés cytocompatibles pour la bioremédiation et la santé
– Biomatériaux pour la réparation oro-faciale

We are seeking a PhD candidate holding a Master’s or Engineering degree in Biomedical Engineering or Materials Chemistry, with a strong background in Cell Biology. A first experience in 3D printing of biopolymers would be an advantage.