Development of new composite scaffolds by freeze-casting for the treatment of the mandibular osteoradionecrosis – CongOs

The work program includes a first step dedicated to the development of nanoprecursors and scaffolds by screening of formulation and process parameters. It is followed by the transfer of technology to Rescoll and the multi-scale (from nanometer to centimeter) and multi-tool physical characterization of the structural, morphological and mechanical properties of scaffolds. The second step is devoted to the biological properties related to the application: i) in vitro studies (biocompatibility, differentiation/proliferation, antibacterial and angiogenic properties) ii) in vitro behavior of the scaffolds implanted in a new animal model adapted to the problems of the MRO. The third step concerns the transfer and evaluation of the industrial potential of the technology.

Five of the six tasks of the project have been started during the first 18 months of the project.
WP1 - Polymers and particle synthesis, functionalization and characterization:
Several functional polymers (mainly based on poly(D,L-lactide)) with different chain lengths have been synthesized in the framework of G. Vecchio's thesis. In connection, several grafting strategies have emerged: i) a «grafting to« strategy based on pre-synthesized polymer grafted to nanoparticles (SiO2-CaO binary nanoparticles previously developed) via a covalent bond (peptide type coupling), ii) a similar «grafting to« strategy but based on a weak interaction (catechol-silanol interaction), iii) a «grafting from« strategy based on the growth of the polymer from the previously grafted nanoparticles, the latter acting as a primer. The syntheses and functionalizations associated with strategies i and ii are mastered and strategy iii is still under optimization. The characterizations associated with the free polymers (NMR, MALDI-TOF) validate the syntheses and those associated with the grafting are still in progress (NMR, FTIR, DLS, TEM) in order to determine the most relevant strategy.
In parallel, work has been carried out on the development of Stöber type syntheses of quaternary nanoparticles (NP) (SiO2-CaO-P2O5-CuO) with a controllable and monodisperse diameter. The integration of copper associated with the obtaining of non-aggregated NPs could finally be achieved through the use of an ionic surfactant. However, the yields and mass associated are low. The generation of small quaternary microparticles by spray-drying has been tested and validated. This process leads to masses compatible with the fabrication of scaffolds and will be optimized during the next months.
The grafting of polymers on this type of particles from the most appropriate strategy will also be implemented.
WP2 - Scaffolds fabrication and their characterization.
First scaffolds have been fabricated combining binary nanoparticles and the ii strategy.
The development of scaffolds combining strategy iii and microparticles generated by spray-drying will be the subject of Gabriele Vecchio's thesis.

WP3 - Multiscale and multi-tool physical characterization of scaffolds:
SAXS beam time has been obtained on the ESRF ID02 line allowing to start this task. About 150 samples of nanoparticles and/or polymers have been analyzed in order to understand the behavior of these nanosystems in suspension and the impact of different parameters: diameter of the nanoparticles, chain length of the grafted polymers, grafting strategy, presence of free polymer, concentration... These measurements have generated a large amount of experimental data which will be processed and analyzed in the framework of the recruitment of a PhD student at IMMM.
The development of a cooled sample holder has allowed to perform in situ aniostropic freezing tests of suspensions.

WP4 - In vitro and in vivo evaluation of biological properties:
Task not started, according to the initial schedule and in view of the progress of the preliminary tasks
WP5 - Technology assessment, transfer and valorization
RESCOLL will participate in the industrial transfer of the synthesis of NPs as well as the implementation by freeze casting of the scaffold developed in the case of the COngOs project. The qualification of the process will also be carried out in order to ensure that the developed product will be in conformity with the GMP. The ISO14971 risk analysis will be carried out after selection of the NP synthesis route.

the synthesis of non-aggregated quaternary nanoparticles (not described in the literature) was developed but required more time than expected (1 L3 and 1 M2 internship). The quantities obtained do not allow to consider the fabrication of scaffolds on a large scale but a conclusive alternative has been found thanks to the use of the atomization-drying process.

Lagarrigue, Prescillia, Vincent Darcos, Christophe Tenailleau, Benjamin Duployer, Agnès Dupret-Bories, Sophie Cazalbou, Dominique Poquillon, David Grossin, Christèle Combes, Jérémy Soulié. « Poly(d,l-lactide)-Grafted Bioactive Glass Nanoparticles: From Nanobricks to Freeze-Cast Scaffolds for Bone Substitution ». ACS Appl. Nano Mater. 2022, 5,278-52912022
doi.org/10.1021/acsanm.2c00313.

Head and neck cancers rank 5th in France and 70% of them are treated with radiotherapy. However some complications can occur, including osteoradionecrosis (ORN) with 5-10% of incidence. This necrosis of the mandibular induces pain, infections, fractures and most of patients can no longer eat orally. While conservative treatments exist for default < 1cm2, no effective treatment/biomaterial is currently available for larger ORN.
The CongOs project intends to develop (from fundamental to applied aspects) a new porous composite material with angiogenic and antibacterial properties for bone reconstruction in ORN site with defect size > 1cm2 and its technological transfer to a SME company. The considered materials are aliphatic polyester/bioactive glass composites scaffolds elaborated by the freeze-casting process. Their elaboration involves well-defined and customized nanoscopic building entities and is based on a new strategy that has never been described for biomedical applications and especially for particles/polymers cited above. This new approach allows obtaining bioactive materials with very high and homogeneous mineral filler rate and modular porosity (volume, pore size, anisotropy similar to mandibular bone) and composition. Then these scaffolds could fulfil the multiple requirements of ORN: i) stimulate the formation of new bone in these critical bone defects thanks to cell differentiation and proliferation. ii) be osteoconductive and exhibit high porosity to support cell colonization and angiogenesis. iv) maintain adequate mechanical properties during handling by surgeons, v) have antibacterial and angiogenic properties. The latter aspect is predominant for ORN. Indeed, even a highly bioactive material will be inefficient to treat ORN after implantation, without addressing the bacterial issue. We propose here a twofold approach in order to avoid it with synergistic and/or dual kinetic/mechanistic effect of several kind of active agents.
Most importantly, this project aims at assessing the composite scaffold technology including the transfer of whole elaboration process (building blocks and scaffolds) and the development of a freeze-casting apparatus prototype for/by the SME company (no commercial apparatus is currently available). To ensure to reach these main R&D objectives, fundamental chemical, physical and biological studies need to be carried out to establish a link between the elaboration process, the scaffolds properties and their biological behaviour.
Therefore, a consortium has been formed with 5 academic and 1 industrial partners, providing complementary domains of knowledge and expertise for the good development of this multidisciplinary project. The consortium includes CIRIMAT in Toulouse, IBMM in Montpellier (both members of Institut Carnot Chimie Balard Cirimat), IUCT in Toulouse, RMeS in Nantes, IMMM in Le Mans and Rescoll in Pessac
The work program includes a first step dedicated to the elaboration of nanoprecursors and scaffolds by screening formulation (compositions of bioactive glasses, polymer chain length, nature of the solvent, concentration…) and process (temperature gradient) parameters. It will be followed by the characterization of structural, morphological and mechanical scaffolds properties and the technology transfer to Rescoll. The second step is dedicated to biological properties correlated with the application: i) in vitro studies such as biocompatibility, differentiation / proliferation of cells and antibacterial and angiogenic properties and ii) in vitro behaviour of implanted scaffolds with a new animal model well-adapted to mimic ORN issues. Finally the thirs step is focused on the transfert and the industrial assesment of the technology.