Conception et caractérisation d'un hydrogel bioactif pour la régénération du disque intervertébral

Abstract

The minimally invasive treatment of intervertebral disc degeneration is an interesting approach to overcome the shortcomings of current surgical techniques. This type of technology requires the encapsulation of cells in injectable scaffolds with or without bioactive factors. The injectable scaffolds will mechanically reinforce the degenerated tissue while the bioactive factors stimulate the encapsulated cells to produce the components necessary for the tissue’s regeneration. In this order, the scaffold must fill both precise mechanical and biological requirements. Given their high water content and their versatility, injectable hydrogels are very interesting candidates. However, the hydrogels currently developed struggle to meet simultaneously the mechanical and biological specifications necessary for their clinical performance. Novel thermoresponsive chitosan hydrogels formulations and a bioactive molecule, Link N respectively developed or evaluated in the laboratories of Professors Sophie Lerouge and Fackson Mwale could overcome these shortcomings. Link N is a bioactive molecule promoting the synthesis of proteoglycans, an important component of the extracellular matrix of the disc responsible for its mechanical properties in compression. The objective of the project is to develop and demonstrate the potential of these hydrogels associated with Link N, to restore the biomechanical properties of the disc, in particular its central part: the nucleus pulposus (NP). The project hypothesizes that these hydrogel formulations, once optimized and associated with Link N, would possess the appropriate mechanical properties and would be suitable for the encapsulation of cells, ensuring their survival as well as the production of an appropriate extracellular matrix. Firstly, an optimization study was carried out to select an optimal formulation consisting of 2% w/v of chitosan, 75 mM of sodium hydrogen carbonate and 100 mM of β glycerophosphate. This formulation is liquid and stable at room temperature and injectable through a needle (22 gauge). It has an adequate gelation rate and mechanical properties in compression and shear close to those of human NP, after complete gelation. The properties of this gel allow encapsulation and survival of NP cells (> 80% viability after 14 days of culture). However, the amount of proteoglycans produced is suboptimal. This proteoglycans production was improved by adding Link N (100µ/mL) and gelatin (1%w/v) in a second step of the project. The efficacy of these bioactive factors has been evaluated and confirmed in a culture medium replicating the biochemical composition of a degenerated disc and therefore indicates the clinical potential of the method. In a third step, ex vivo tests in a degenerated bovine disc model were carried out to evaluate the capacity of the gel to restore the mechanical properties of the disc. However, the diffusion of the degeneration solution outside the NP towards the rest of the disc prevented us from going through with this step. Nevertheless, a protocol has been developed to define the important parameters to be considered to reliably perform this type of study. Finally, to assess the potential for clinical transfer of the technology, the viability of mesenchymal stem/stromal cells (MSC) encapsulated in this gel was studied. These results show that the sensitivity of these cells and the lack of cell adhesion patterns in the chitosan gel induce a low cell viability for encapsulated MSC. Therefore, the technology remains to be improved to increase the viability of MSCs which are the most appropriate cells for clinical application of this technology.

Date	26 Aug 2022
Original language	French
Awarding Institution	École de technologie supérieure
Supervisor	Sophie Lerouge (Supervisor) & Fackson Mwale (Co-supervisor)