Oriented supramolecular gels as a guide for neural stem cells
Cerebrovascular stroke or trauma lead to an important loss of neuronal tissue, leading to strong disability. A current challenge in the field of tissue engineering is to make implants that first ensure a good survival of implanted neuronal cells. A greater challenge is also to direct the growth of these neuronal cells in a preferential direction, in order to organize the tissue reconstruction and to reconnect the parts separated by the damage. The objective of the project is to prepare and to inject in vivo biocompatible and oriented gels charged with human neural stem cells. The aim is to demonstrate that these gels will be able to direct the growth of neurons in a preferential direction and therefore will improve the motor functional recovery and quality.
Three main skills were implemented. The starting point is the design and synthesis of a family of molecules that self-assemble to form fibers, supporting the formation of gels. The gels have been used as scaffolds for neuron cell culture. The biocompatibility has been assessed by performing viability tests on neural cell lines. The growth in 3D, within the molecular gel fibrillar network has been evidenced by confocal microscopy. Then, human adult neural stem cell culture was performed. Methods have been specifically developed to successfully observe and identify cells in these very fragile gels. Finally, various methods for shaping the gels have been implemented, including wet spinning and 3D printing.
A family of fourteen carbohydrate derived gelators has been synthesized. Gelling conditions and hydrogels were characterized by different physicochemical methods. The biocompatibility of seven of them has been evaluated, with good results in terms of cell viability. Culture of adult human neural stem cells on the newly developed hydrogels showed that stem cells develop in seven days into a dense network of neurons and glial cells. In addition, neurons develop numerous neurites guided by molecular gel fibers. In particular, the formation of a mixed network of neurites was observed, with bundles of very long rectilinear neurites following the straight and wide fibers of the gelling agent and other shorter neurites of non-rectilinear morphology. Then, we developed a method for shaping these hydrogels by extrusion. The injection of a solution of gelling agent in water causes it to coagulate on contact with water. Depending on the set-up, coils of gel filaments are formed, by wet spinning or small 3D printed architectures are formed, by 3D printing. This technique makes it possible to shape the gelling agents very precisely and more easily than by thermal means. In addition, remarkably, during wet spinning, the supramolecular gelling fibers have organized radially within the gel filament. This particular organization reveals the radial diffusion of water during the gelling process.
The family of carbohydrate gelators and the shaping techniques developed in this project opens up very interesting perspectives in the field of materials and hydrogels as scaffolds for cell culture. Indeed, they are synthetic, pure molecules, with perfectly defined, reproducible structures and biocompatible. In addition, a 3D printing method makes it possible to build very precise architectures with these hydrogels. These new molecules therefore present important perspectives in the field of 3D bioprinting and tissue engineering.
1- «A Shear-Induced Network of Aligned Wormlike Micelles in a Sugar-Based Molecular Gel. From Gelation to Biocompatibility Assays«. J. Fitremann, B. Lonetti, E. Fratini, I. Fabing, B. Payré, C. Boulé, I. Loubinoux, L. Vaysse, L. Oriol, Journal of Colloid and Interface Science, 2017, 504, 721–730.
2- «Simple synthetic molecular hydrogels from self-assembling alkylgalactonamides as scaffold for 3D neuronal cell growth«. A. Chalard, L. Vaysse, P. Joseph, L. Malaquin, S.Souleille, B. Lonetti, J.-C. Sol, I. Loubinoux, J. Fitremann, ACS Applied Materials and Interfaces, 2018, 10, 17004-17017.
3- Chalard, A.; Joseph, P.; Souleille, S.; Lonetti, B.; Saffon-Merceron, N.; Loubinoux, I.; Vaysse, L.; Malaquin, L.; Fitremann, J. Wet Spinning and Radial Self-Assembly of a Carbohydrate Low Molecular Weight Gelator into Well Organized Hydrogel Filaments. Nanoscale 2019, 11 (32), 15043–15056.
4- Chalard, A.; Mauduit, M. ; Souleille, S.; Joseph, P.; Malaquin, L.; Fitremann, J. 3D printing of a biocompatible low molecular weight supramolecular hydrogel by dimethylsulfoxide - water solvent exchange. Additive Manufacturing 2020, 33, 101162.
5- Production of N-heptyl-D-galactonamide, biological grade (licence)
6. CNRS national press release «A new gelling molecule for growing neurons in 3D«, 14 mai 2018.
15 oral communications in national and international congresses
Cerebrovascular stroke or trauma lead to an important loss of neuronal tissue, leading to strong disability. A current challenge in the field of tissue engineering is to make implants which first ensure a good survival of implanted neuronal cells and furthermore, which allow to direct growth of these neuronal cells in a preferential direction, in order to organize the tissue reconstruction and to reconnect the two parts separated by the lesion. The objective of the project is to prepare and to inject in vivo biocompatible oriented gels charged with human neural stem cells. The aim is to demonstrate that these gels will guide the growth of neurons in a preferential direction and that they can improve the motor functional recovery and quality.
For this purpose, a first step is to design and prepare new hydrogels for 3D cell culture, based on "Low Molecular Weight" (LMW) supramolecular hydrogelators. These gelators tend to form by self-assembly entangled fibers in water which support the formation of viscoelastic hydrogels. Their mechanical and rheological properties differ from polymer based hydrogels and can better mimic the properties of the extracellular matrix. In some cases, they also display fast gel recovery after shear as can do "self-healing" materials. For this reason, they could better fulfill the rheological requirements in their application as injectable matrices and could be more suitable for 3D cell cultures compared with polymer gels. Compared with few other LMW hydrogels already traded for 3D cell culture and injectability, based on quite expensive peptides, the objective is to design and synthetize of biocompatible "Low Molecular Weight" supramolecular hydrogelators belonging to other families of molecules. We will determine their biocompatibility and their rheological properties in relation with their use as in vitro 3D cell culture matrix and as injectable matrix for in vivo applications.
In order to induce the oriented growth of neuronal cells, different methods for orienting the supramolecular fibers will be implemented. The objective is to better control the self-assembly of the "dissociated molecules" to fibers to get well-defined and well-organized fibrillar aggregates, and to get more particularly oriented fibers. The design of specific devices for controlling the alignment and the gelation triggers will be implemented.
Finally, 3D in vitro neural cell cultures, including human neural stem cells, will be performed on the hydrogels, with or without orientation. Cytotoxicity, growth, adhesion and cell differentiation will be studied. Finally, the ability of hydrogels oriented to guide the growth of neurites in one preferred direction will be assessed. The final achievement will be to test, with the gels giving the best results in vitro, whether they can be injected and oriented in vivo. The results will be studied from the point of view of functional recovery and tissue reconstruction.
Madame Juliette FITREMANN (Centre National de la Recherche Scientifique/Interactions Moléculaires et Réactivité Chimique et Photochimique)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
INSERM/ICHN/U825 Institut national de la santé et de la recherche médicale/Imagerie Cérébrale et Handicaps Neurologiques
CNRS/LAAS-CNRS Centre National de la Recherche Scientifique/Laboratoire d'Analyse et d'Architecture des Systèmes
CNRS/IMRCP Centre National de la Recherche Scientifique/Interactions Moléculaires et Réactivité Chimique et Photochimique
Help of the ANR 412,131 euros
Beginning and duration of the scientific project: December 2015 - 48 Months