Self-assembled printable hydrogels from biocompatible amphiphiles for tissue engineering – SELFAMPHI

First, we tested the hydrogelation properties of many bio-based amphiphiles, the objective being to find the optimal conditions to form hydrogels in various aqueous media, to understand their structure and formation, to establish a reproducible protocol of their synthesis, to test the effect of physico-chemical parameters and to characterize their preliminary mechanical properties. We used rheology, small-angle X-ray scattering (SAXS) and rheology coupled with SAXS to study the properties and structure of hydrogels.

Next, we investigated the flow of hydrogels and possible 3D printing. Our first objective is to understand the flow, fluidization and gelation mechanisms of these hydrogels. The reactivity to stimuli (pH, saline, thermal, photonics, etc.) was tested in-situ as well as the control of critical process parameters (drying, crosslinking, thixotropy, fluidization under flow, etc.). The mechanical strength, resolution, adhesion and temporal stability of the structures must be optimized. The 3D printing was also tested following the validation of the fluidization properties.

Ultimately, cytotoxicity in the presence of NHDF cells was tested for molecules producing stable hydrogels.

At LCMCP in Paris, we found that two molecules form gels in a reproducible way. The former forms hydrogels with a basic pH above 7.5. The gels have significant mechanical properties, with elastic moduli of the order of kPa, or even more depending on the concentration (up to 5 wt%). The gels are stable over time and in temperature up to 60-80°C, depending on the system. We studied the structure of the gels and the result is an unexpected fibrillar system. We have identified two mechanisms underlying the formation of gels. We have also identified another system that forms hydrogels according to the temperature below 30°C, which is quite interesting for 3D printing.

The study of the mechanisms was carried out by combining several techniques on different synchrotrons (SOLEIL, ESRF). We carried out several sessions, some of which combined rheology and SAXS.

The LRP in Grenoble then conducted an in-depth analysis of the rheological properties of the hydrogels using various rheometric tools. The work evaluated the viscoelastic properties, rheofluidification, flow thresholds and thixotropy of hydrogels over a wide range of compositions, concentrations and preparation methods. This research has made it possible to optimize the formulation and preparation conditions for optimal 3D printability, while demonstrating the importance of the aging of hydrogels on their stability and behavior in 3D printing.

Cytotoxicity measurements were performed with all microbial surfactants studied. Significant cytotoxicity was observed at all treatments at concentrations greater than 1% (w/v) after only 24 hours of culture. In contrast, no cytotoxicity was observed at concentrations below 0.01% (w/v). In the intermediate concentration range (1-0.01% w/v), significant cytotoxicity was observed for only some molecules. The complementary cytotoxicity assessment was performed on NHDF cells treated for 24 and 48 hours. The work shows that the molecules are cytotoxic to NHDF cells at concentrations above 1% w/v. It should be noted that non-cytotoxic concentrations are below the concentrations of surfactants that can form gels. This limited the rest of the study and opens up prospects for the use of these molecules in composite form.

This work has mainly explored the glycolipid family. However, other bioamphiphiles, such as cyclic peptides, exist. It would be very interesting to know their hydrogel-forming properties. Indeed, peptides may have interesting adhesion and biomolecular recognition properties, which means that amphiphilic hydrogels of microbial peptides could have even greater potential in the biomedical field. At present, there is no information on the potential hydrogelation of microbial peptide amphiphiles.

Regarding the properties of the hydrogels discovered during this project, it would be interesting to increase their elastic properties, for example by integrating reinforcing agents such as cellulose nanocrystals or by developing mixed interpenetrated networks with biopolymers. This approach would reduce the initial concentration of low-molecular weight gelators, as well as the time required for maturation and stabilization of interactions, while obtaining printable materials with improved mechanical properties. Another perspective is to optimize the process to make it possible to print structures directly in situ, ensuring greater homogeneity and efficiency.

Ultimately, given the potential toxicity of hydrogels made from the bio-based amphiphiles studied in this project, it will first be necessary to study the in vitro degradability of the hydrogels and test the toxicity of the molecules released. An in vivo study may also be carried out in rats. To do this, a subcutaneous implantation will have to be performed and the inflammation around the implant studied. The development of reinforced or interpenetrated hydrogels would also be an interesting approach to reduce the intrinsic cytotoxicity of the matrix.

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Biological surfactants are a family of natural molecules obtained from the microbial digestion of fatty acids and sugars. These compounds are developed the low impact of the synthesis process (industrial biotechnology), low toxicity and high biodegradability. Sophorolipids, glucolipids or cellobioselipids are some of the most important molecules. Historically developed as biodegradable detergents to replace petrochemicals, their high cost/benefit ratio stimulates their use for other high-end applications. Recent work demonstrates their self-assembly properties into fibers and bilayers, which show the formation of shear-thinning stimuli-responsive hydrogels. This class of soft materials is an interesting alternative to known matrices for tissue engineering, a field in continuous seek for new biomaterials due to problems of contamination, purity, cell adhesion and cost. The goal of SELFAMPHI is to use biocompatible amphiphiles to develop new injectable and printable hydrogels to test in tissue engineering (spinal disc repair and skin fillers) applications.